Genetic and enzymatic characterization of 3-O-sulfotransferase SNPs associated with Plasmodium falciparum parasitaemia

Genetic and enzymatic characterization of 3-O-sulfotransferase SNPs associated with Plasmodium... Abstract The HS3ST3A1/B1 genes encode two homologous 3-O-sulfotransferases involved in the late modification step during heparan sulfate (HS) biosynthesis. In addition to the single nucleotide polymorphisms (SNPs) rs28470223 (C > T) in the promoter region of both HS3ST3A1 and rs62636623 (Gly/Arg) in the stem region of HS3ST3B1, three missense mutations (rs62056073, rs61729712 and rs9906590) located within the catalytic sulfotransferase domain of 3-OST-B1 are linked and associated to Plasmodium falciparum parasitaemia. To ascertain the functional effects of these SNP associations, we investigated the regulatory effect of rs28470223 and characterized the enzymatic activity of the missense SNP rs61729712 (Ser279Asn) localized at proximity of the substrate binding cleft. The SNP rs28470223 results in decreased promoter activity of HS3ST3A1 in K562 cells, suggesting a reduced in vivo transcription activity of the target gene. A comparative kinetic analysis of wt HS3ST3B1 and the Ser269Asn variant (rs61729712) using a HS-derived oligosaccharide substrate reveals a slightly higher catalytic activity for the SNP variant. These genetic and enzymatic studies suggest that genetic variations in enzymes responsible of HS 3-O-sulfation can modulate their promoter and enzymatic activities and may influence P. falciparum parasitaemia. enzymatic activity, genetic association, heparan sulfate biosynthesis, 3-O-sulfotransferase Introduction Heparan sulfate (HS) is a family of highly N- and O-sulfated glycosaminoglycans that is commonly found on the mammalian cell surface and in the extracellular matrix. HS is attached to a variety of different core proteins defined as HS proteoglycans (HSPGs) and participates in a wide range of physiological and pathophysiological functions, including embryonic development, inflammatory responses, blood coagulation and assisting viral/bacterial infections (Bishop et al. 2007). HS consists of a disaccharide repeating unit with D-glucuronic acid (GlcA) or L-iduronic acid (IdoA) and glucosamine, each of which is capable of carrying sulfate groups and contributes to dictate HS function (Bishop et al. 2007). The HS maturation process involves a series of biosynthetic enzymes, including bifunctional enzymes (NSDT, N-deacetylase/N-sulfotransferase), a critical step for formation of N-sulfated domains, a C5-epimerase and various O-sulfotransferases (Fu et al. 2016). The three classes of O-sulfotransferases transfer sulfate groups at various positions, including C2 of IdoA, C6 and C3 of the GlcN units, leading to clusters of highly sulfated regions (NS domain) separated by long stretches of unsulfated GlcNAc-containing disaccharides (NA domain). The sulfate-rich regions are likely to generate highly flexible regions because of their high content in the conformationally versatile IdoA and IdoA2S residues (Turnbull and Gallagher 1991). In addition, tissue specific expression of different enzyme isoforms fine-tunes synthesis of specific HS structures, which are predominantly determined by a controlled positioning of N-, 2-, 6- and 3-O-sulfate groups along HS chains, to confer an important regulatory role in various biological processes (Esko and Selleck 2002). The 3-O-sulfation of GlcN unit is a relatively rare modification, present in only a limited number of HS polymers or absent entirely, and occur at a late step during the biosynthetic pathway (Thacker et al. 2014). In this context, the natural abundance of HS 3-O-sulfation may be re-evaluated given the occurrence of a peeling reaction that specifically degrades 3-O-sulfated GlcN unit at the reducing ends of enzymatically depolymerized HS/heparin chains (Huang et al. 2015). Conversely, the HS 3-O-sulfotransferases (3-OST-3 that are encoded by HS3ST3 genes) represent the largest gene family among all HS sulfotransferases and vertebrates generally possess seven isozymes divided into two subgroups according to sequence homology of their sulfotransferase domain (Liu and Pedersen 2007). Based on the large diversity of 3-OST enzymes and the fact that they probably act at a late stage, one might assume that they show selectivity for the sulfation pattern in their target sites. One group (3-OST-2,-3A,-3B,-4 and -6) is often referred to as “gD-type” HS3STs, because members of the subfamily can generate binding sites for glycoprotein gD of type I herpes simplex virus (HSV-1). Members of the second group (3-OST-1, -5) have in common the capacity to generate a binding site for antithrombin and are thus designed “AT-type” HS3STs. Hence, 3-OST-1 preferentially modifies GlcN sites in which GlcA at position +1 is devoid of 2-O-sulfate groups (Mochizuki et al. 2008). In contrast, 3-OST-2, -3, -4 and -6 (gD-type) preferentially modify GlcN sites in which position -1 is IdoA2S (Meissen et al. 2009). Unlike 3-OST-1, the 3-OST-2, -3, -3A and -3B enzymes are predicted to have type II integral membrane architecture (Shworak et al. 1999). Both mouse and human forms of 3-OST-3B and a human form of 3-OST-3A can modify the HS of HSV-1-resistant cells at specific sites to generate cells susceptible to HSV-1 infection (Shukla et al. 1999). In addition to their functional implications in various cell events, HS proteoglycans can play an important role in infectious diseases, such as malaria caused by the parasite Plasmodium, involving the HS chains of both the mammalian host and the vector. Several studies suggest that the outcome of malaria infection may be influenced by differences in HS composition, owing to genetic variations within genes encoding the biosynthesis enzymes (Coppi et al. 2007; Sinnis et al. 2007; Armistead et al. 2011). The genes encoding the 3-O-sulfotransferases are located in different chromosomal regions except for HS3ST3A1 and HS3ST3B1 which encode the 3-OST-3A and 3-OST-3B sulfotransferases, respectively, and target the same disaccharide (Liu, Shriver, et al. 1999). In fact, those two genes are located within chromosome 17p12 that showed a genome-wide significant linkage with Plasmodium falciparum parasitaemia in a population living in Burkina Faso (Brisebarre et al. 2014). Moreover, genetic variations within HS3ST3A1 and HS3ST3B1 are linked to P. falciparum parasitaemia in humans, as found in a population living in an endemic area in Burkina Faso (Atkinson et al. 2012). Alteration of HS 3-O-sulfation may affect (i) the binding of P. falciparum antigen on host cells and/or (ii) the pro-inflammatory response. To date, several single nucleotide polymorphisms (SNPs) have been identified in the promoter region of both HS3ST3A1/B1, and one of them (rs28470223, C/T), which is located within the promoter of HS3ST3A1, was linked and associated with P. falciparum parasitaemia. Moreover, four missense SNPs (rs62636623 G > C Gly83Arg, rs62056073 A > G Ile196Val, rs61729712 G > A Ser269Asn and rs9906590 G > A Glu363Lys) are located within HS3ST3B1, of which the later three SNPs are located within the sulfotransferase domain. These missense SNPs were genetically linked to parasitaemia and were associated with parasitaemia in combination with other SNPs, suggesting that they may alter 3-OST-3 functionality. While epidemiological correlation studies relate naturally occurring variations in 3-O-sulfotransferases to the susceptibility for infectious diseases in these populations, no functional data are yet available for most of these genetic variants. To ascertain the functional role of the SNPs localized either in the promotor region or within the sulfotransferase domain of the HS 3-OST-3 modification enzyme, we investigated the effect of the SNP rs28470223 on the expression level of the HS3ST3A1 gene and the effect of the Ser269Asn (rs61729712) variant on the enzymatic activity of human 3-OST-3B1. Bioinformatics analyses reveal that seven SNPs in HS3ST3A1/B1 show a predicted deleterious effect, confirming that genetic variations within the genes encoding 3-O-sulfotransferases may affect the susceptibility to infectious diseases, such as malaria. The SNP rs28470223 is associated with a significantly altered promoter activity that may affect both the transcriptional activity and P. falciparum parasitaemia in humans. Complementary enzyme kinetic analyses of wt human 3-OST-3 and the Ser269Asn (rs61729712) variant, which is located at the entry of the substrate binding cleft based on the crystal structure of the homologous 3-OST-3A1 isoform, reveal a slightly higher catalytic activity compared with the wt enzyme. Results and discussion Bioinformatics analysis of the phenotypic effect of HS3ST3A1 and HS3ST3B1 genetic variants All SNPs previously identified in the chromosomal region containing HS3ST3A1 and HS3ST3B1 from a population living in an endemic area in Burkina Faso (Atkinson et al. 2012) were investigated using the bioinformatics PredictSNP2 tool, which integrates binary predictions and uniform confidence values computed by six different tools. The comparative analysis, which provides a consensus score based on the five best-performing tools, reveals that seven SNPs show a predicted deleterious effect, including three (rs62056073 A > G Ile196Val, rs61729712 G > A Ser269Asn and rs9906590 G > A Glu363Lys) out of the four missense variants (Table I). In contrast, rs28470223 C > T could not be predicted for deleterious effects using the PredictSNP2 tool in contrast to the alternative FunSeq2 and GWAVA predicting tools. Table I. Predicted effect for HS3ST3A1 and HS3ST3B1 variants SNP_ID  GRCh37 location (bp)  Variant  Gene location  Type of change  PredictSNP2  CADD  DANN  FATHMM  FunSeq2  GWAVA  rs62057033  13399616  A > T  HS3ST3A1  Exon 2 - Synonymous  96% Neutral  73% Neutral  90% Neutral  77% Neutral  93% Neutral  56% Neutral  rs61732181  13399928  C > T  HS3ST3A1  Exon 2 - Synonymous  88% Neutral  58% Deleterious  90% Neutral  93% Deleterious  93% Neutral  56% Neutral  rs61744056  13400057  T > C  HS3ST3A1  Exon 2 - Synonymous  91% Neutral  94% Neutral  97% Neutral  90% Neutral  93% Neutral  62% Deleterious  rs8080565  13400153  G > A  HS3ST3A1  Intron  77% Neutral  86% Neutral  54% Deleterious  85% Neutral  80% Neutral  58% Neutral  rs3744337  13504665  A > G  HS3ST3A1  5′UTR  74% Neutral  67% Deleterious  62% Neutral  93% Neutral  83% Neutral  64% Deleterious  rs3744335  13504884  T > G  HS3ST3A1  5′UTR  88% Neutral  86% Neutral  80% Neutral  93% Neutral    53% Neutral  rs28470223  13505023  A > G  HS3ST3A1  5′UTR  88% Neutral  76% Neutral  86% Neutral  91% Neutral  67% Deleterious  64% Deleterious  rs78863672  13505237  C > A  HS3ST3A1  5′UTR  91% Deleterious  86% Deleterious  62% Neutral  86% Deleterious  67% Deleterious  84% Deleterious  rs2072243  14204380  C > T  HS3ST3B1  5′UTR  97% Deleterious  76% Deleterious  89% Deleterious    68% Deleterious  80% Deleterious  rs2072242  14204410  T > C  HS3ST3B1  5′UTR  97% Deleterious  98% Deleterious  89% Deleterious  88% Deleterious  62% Deleterious  78% Deleterious  rs115229628  14204423  G > A  HS3ST3B1  5′UTR  97% Deleterious  98% Deleterious  100% Deleterious  91% Deleterious  67% Deleterious  76% Deleterious  rs62636623  14205082  G > C  HS3ST3B1  Exon 1 - Missense Gly > Arg  89% Neutral  94% Neutral  90% Neutral  77% Neutral  84% Neutral    rs62636622  14205168  G > A  HS3ST3B1  Exon 1 - Synonymous  96% Neutral  92% Neutral  90% Neutral  87% Neutral  93% Neutral  54% Neutral  rs62056073  14248376  A > G  HS3ST3B1  Exon 2 - Missense Ile > Val  87% Deleterious  58% Neutral  60% Deleterious  59% Deleterious  62% Neutral  51% Deleterious  rs9906855  14248423  T > C  HS3ST3B1  Exon 2 - Synonymous  96% Neutral  94% Neutral  97% Neutral  90% Neutral  93% Neutral  62% Deleterious  rs61729712  14248596  G > A  HS3ST3B1  Exon 2 - Missense Ser>Asn  87% Deleterious  53% Deleterious  60% Deleterious  82% Deleterious  62% Neutral  51% Deleterious  rs55688668  14248702  G > A  HS3ST3B1  Exon 2 - Synonymous  93% Neutral  83% Neutral  90% Neutral  57% Deleterious  93% Neutral  56% Neutral  rs9906590  14248877  G > A  HS3ST3B1  Exon 2 - Missense Glu > Lys  87% Deleterious  53% Deleterious  60% Deleterious  65% Deleterious  62% Neutral  50% Deleterious  rs3785655  14249167  C > T  HS3ST3B1  3′UTR  88% Neutral  86% Neutral  68% Neutral  75% Neutral  83% Neutral  53% Neutral  rs73979332  14249433  C > T  HS3ST3B1  3′UTR  88% Neutral  80% Neutral  62% Neutral  89% Neutral  81% Neutral  71% Neutral  SNP_ID  GRCh37 location (bp)  Variant  Gene location  Type of change  PredictSNP2  CADD  DANN  FATHMM  FunSeq2  GWAVA  rs62057033  13399616  A > T  HS3ST3A1  Exon 2 - Synonymous  96% Neutral  73% Neutral  90% Neutral  77% Neutral  93% Neutral  56% Neutral  rs61732181  13399928  C > T  HS3ST3A1  Exon 2 - Synonymous  88% Neutral  58% Deleterious  90% Neutral  93% Deleterious  93% Neutral  56% Neutral  rs61744056  13400057  T > C  HS3ST3A1  Exon 2 - Synonymous  91% Neutral  94% Neutral  97% Neutral  90% Neutral  93% Neutral  62% Deleterious  rs8080565  13400153  G > A  HS3ST3A1  Intron  77% Neutral  86% Neutral  54% Deleterious  85% Neutral  80% Neutral  58% Neutral  rs3744337  13504665  A > G  HS3ST3A1  5′UTR  74% Neutral  67% Deleterious  62% Neutral  93% Neutral  83% Neutral  64% Deleterious  rs3744335  13504884  T > G  HS3ST3A1  5′UTR  88% Neutral  86% Neutral  80% Neutral  93% Neutral    53% Neutral  rs28470223  13505023  A > G  HS3ST3A1  5′UTR  88% Neutral  76% Neutral  86% Neutral  91% Neutral  67% Deleterious  64% Deleterious  rs78863672  13505237  C > A  HS3ST3A1  5′UTR  91% Deleterious  86% Deleterious  62% Neutral  86% Deleterious  67% Deleterious  84% Deleterious  rs2072243  14204380  C > T  HS3ST3B1  5′UTR  97% Deleterious  76% Deleterious  89% Deleterious    68% Deleterious  80% Deleterious  rs2072242  14204410  T > C  HS3ST3B1  5′UTR  97% Deleterious  98% Deleterious  89% Deleterious  88% Deleterious  62% Deleterious  78% Deleterious  rs115229628  14204423  G > A  HS3ST3B1  5′UTR  97% Deleterious  98% Deleterious  100% Deleterious  91% Deleterious  67% Deleterious  76% Deleterious  rs62636623  14205082  G > C  HS3ST3B1  Exon 1 - Missense Gly > Arg  89% Neutral  94% Neutral  90% Neutral  77% Neutral  84% Neutral    rs62636622  14205168  G > A  HS3ST3B1  Exon 1 - Synonymous  96% Neutral  92% Neutral  90% Neutral  87% Neutral  93% Neutral  54% Neutral  rs62056073  14248376  A > G  HS3ST3B1  Exon 2 - Missense Ile > Val  87% Deleterious  58% Neutral  60% Deleterious  59% Deleterious  62% Neutral  51% Deleterious  rs9906855  14248423  T > C  HS3ST3B1  Exon 2 - Synonymous  96% Neutral  94% Neutral  97% Neutral  90% Neutral  93% Neutral  62% Deleterious  rs61729712  14248596  G > A  HS3ST3B1  Exon 2 - Missense Ser>Asn  87% Deleterious  53% Deleterious  60% Deleterious  82% Deleterious  62% Neutral  51% Deleterious  rs55688668  14248702  G > A  HS3ST3B1  Exon 2 - Synonymous  93% Neutral  83% Neutral  90% Neutral  57% Deleterious  93% Neutral  56% Neutral  rs9906590  14248877  G > A  HS3ST3B1  Exon 2 - Missense Glu > Lys  87% Deleterious  53% Deleterious  60% Deleterious  65% Deleterious  62% Neutral  50% Deleterious  rs3785655  14249167  C > T  HS3ST3B1  3′UTR  88% Neutral  86% Neutral  68% Neutral  75% Neutral  83% Neutral  53% Neutral  rs73979332  14249433  C > T  HS3ST3B1  3′UTR  88% Neutral  80% Neutral  62% Neutral  89% Neutral  81% Neutral  71% Neutral  Table I. Predicted effect for HS3ST3A1 and HS3ST3B1 variants SNP_ID  GRCh37 location (bp)  Variant  Gene location  Type of change  PredictSNP2  CADD  DANN  FATHMM  FunSeq2  GWAVA  rs62057033  13399616  A > T  HS3ST3A1  Exon 2 - Synonymous  96% Neutral  73% Neutral  90% Neutral  77% Neutral  93% Neutral  56% Neutral  rs61732181  13399928  C > T  HS3ST3A1  Exon 2 - Synonymous  88% Neutral  58% Deleterious  90% Neutral  93% Deleterious  93% Neutral  56% Neutral  rs61744056  13400057  T > C  HS3ST3A1  Exon 2 - Synonymous  91% Neutral  94% Neutral  97% Neutral  90% Neutral  93% Neutral  62% Deleterious  rs8080565  13400153  G > A  HS3ST3A1  Intron  77% Neutral  86% Neutral  54% Deleterious  85% Neutral  80% Neutral  58% Neutral  rs3744337  13504665  A > G  HS3ST3A1  5′UTR  74% Neutral  67% Deleterious  62% Neutral  93% Neutral  83% Neutral  64% Deleterious  rs3744335  13504884  T > G  HS3ST3A1  5′UTR  88% Neutral  86% Neutral  80% Neutral  93% Neutral    53% Neutral  rs28470223  13505023  A > G  HS3ST3A1  5′UTR  88% Neutral  76% Neutral  86% Neutral  91% Neutral  67% Deleterious  64% Deleterious  rs78863672  13505237  C > A  HS3ST3A1  5′UTR  91% Deleterious  86% Deleterious  62% Neutral  86% Deleterious  67% Deleterious  84% Deleterious  rs2072243  14204380  C > T  HS3ST3B1  5′UTR  97% Deleterious  76% Deleterious  89% Deleterious    68% Deleterious  80% Deleterious  rs2072242  14204410  T > C  HS3ST3B1  5′UTR  97% Deleterious  98% Deleterious  89% Deleterious  88% Deleterious  62% Deleterious  78% Deleterious  rs115229628  14204423  G > A  HS3ST3B1  5′UTR  97% Deleterious  98% Deleterious  100% Deleterious  91% Deleterious  67% Deleterious  76% Deleterious  rs62636623  14205082  G > C  HS3ST3B1  Exon 1 - Missense Gly > Arg  89% Neutral  94% Neutral  90% Neutral  77% Neutral  84% Neutral    rs62636622  14205168  G > A  HS3ST3B1  Exon 1 - Synonymous  96% Neutral  92% Neutral  90% Neutral  87% Neutral  93% Neutral  54% Neutral  rs62056073  14248376  A > G  HS3ST3B1  Exon 2 - Missense Ile > Val  87% Deleterious  58% Neutral  60% Deleterious  59% Deleterious  62% Neutral  51% Deleterious  rs9906855  14248423  T > C  HS3ST3B1  Exon 2 - Synonymous  96% Neutral  94% Neutral  97% Neutral  90% Neutral  93% Neutral  62% Deleterious  rs61729712  14248596  G > A  HS3ST3B1  Exon 2 - Missense Ser>Asn  87% Deleterious  53% Deleterious  60% Deleterious  82% Deleterious  62% Neutral  51% Deleterious  rs55688668  14248702  G > A  HS3ST3B1  Exon 2 - Synonymous  93% Neutral  83% Neutral  90% Neutral  57% Deleterious  93% Neutral  56% Neutral  rs9906590  14248877  G > A  HS3ST3B1  Exon 2 - Missense Glu > Lys  87% Deleterious  53% Deleterious  60% Deleterious  65% Deleterious  62% Neutral  50% Deleterious  rs3785655  14249167  C > T  HS3ST3B1  3′UTR  88% Neutral  86% Neutral  68% Neutral  75% Neutral  83% Neutral  53% Neutral  rs73979332  14249433  C > T  HS3ST3B1  3′UTR  88% Neutral  80% Neutral  62% Neutral  89% Neutral  81% Neutral  71% Neutral  SNP_ID  GRCh37 location (bp)  Variant  Gene location  Type of change  PredictSNP2  CADD  DANN  FATHMM  FunSeq2  GWAVA  rs62057033  13399616  A > T  HS3ST3A1  Exon 2 - Synonymous  96% Neutral  73% Neutral  90% Neutral  77% Neutral  93% Neutral  56% Neutral  rs61732181  13399928  C > T  HS3ST3A1  Exon 2 - Synonymous  88% Neutral  58% Deleterious  90% Neutral  93% Deleterious  93% Neutral  56% Neutral  rs61744056  13400057  T > C  HS3ST3A1  Exon 2 - Synonymous  91% Neutral  94% Neutral  97% Neutral  90% Neutral  93% Neutral  62% Deleterious  rs8080565  13400153  G > A  HS3ST3A1  Intron  77% Neutral  86% Neutral  54% Deleterious  85% Neutral  80% Neutral  58% Neutral  rs3744337  13504665  A > G  HS3ST3A1  5′UTR  74% Neutral  67% Deleterious  62% Neutral  93% Neutral  83% Neutral  64% Deleterious  rs3744335  13504884  T > G  HS3ST3A1  5′UTR  88% Neutral  86% Neutral  80% Neutral  93% Neutral    53% Neutral  rs28470223  13505023  A > G  HS3ST3A1  5′UTR  88% Neutral  76% Neutral  86% Neutral  91% Neutral  67% Deleterious  64% Deleterious  rs78863672  13505237  C > A  HS3ST3A1  5′UTR  91% Deleterious  86% Deleterious  62% Neutral  86% Deleterious  67% Deleterious  84% Deleterious  rs2072243  14204380  C > T  HS3ST3B1  5′UTR  97% Deleterious  76% Deleterious  89% Deleterious    68% Deleterious  80% Deleterious  rs2072242  14204410  T > C  HS3ST3B1  5′UTR  97% Deleterious  98% Deleterious  89% Deleterious  88% Deleterious  62% Deleterious  78% Deleterious  rs115229628  14204423  G > A  HS3ST3B1  5′UTR  97% Deleterious  98% Deleterious  100% Deleterious  91% Deleterious  67% Deleterious  76% Deleterious  rs62636623  14205082  G > C  HS3ST3B1  Exon 1 - Missense Gly > Arg  89% Neutral  94% Neutral  90% Neutral  77% Neutral  84% Neutral    rs62636622  14205168  G > A  HS3ST3B1  Exon 1 - Synonymous  96% Neutral  92% Neutral  90% Neutral  87% Neutral  93% Neutral  54% Neutral  rs62056073  14248376  A > G  HS3ST3B1  Exon 2 - Missense Ile > Val  87% Deleterious  58% Neutral  60% Deleterious  59% Deleterious  62% Neutral  51% Deleterious  rs9906855  14248423  T > C  HS3ST3B1  Exon 2 - Synonymous  96% Neutral  94% Neutral  97% Neutral  90% Neutral  93% Neutral  62% Deleterious  rs61729712  14248596  G > A  HS3ST3B1  Exon 2 - Missense Ser>Asn  87% Deleterious  53% Deleterious  60% Deleterious  82% Deleterious  62% Neutral  51% Deleterious  rs55688668  14248702  G > A  HS3ST3B1  Exon 2 - Synonymous  93% Neutral  83% Neutral  90% Neutral  57% Deleterious  93% Neutral  56% Neutral  rs9906590  14248877  G > A  HS3ST3B1  Exon 2 - Missense Glu > Lys  87% Deleterious  53% Deleterious  60% Deleterious  65% Deleterious  62% Neutral  50% Deleterious  rs3785655  14249167  C > T  HS3ST3B1  3′UTR  88% Neutral  86% Neutral  68% Neutral  75% Neutral  83% Neutral  53% Neutral  rs73979332  14249433  C > T  HS3ST3B1  3′UTR  88% Neutral  80% Neutral  62% Neutral  89% Neutral  81% Neutral  71% Neutral  SNP rs28470223 regulates transcriptional promoter activity of HS3ST3A1 We further used bioinformatics to investigate the regulatory effect of the SNP rs28470223 (C > T) associated with a malaria phenotype (Atkinson et al. 2012). The location of rs28470223 within the promoter suggested that this SNP is a cis-regulatory variant controlling HS3ST3A1 gene expression. We first mapped the peaks from ChIP-seq experiments covering a total of 485 transcription factors using the Remap atlas (Griffon et al. 2015; Chèneby et al. 2018) and found 56 transcription factors that bind to rs28470223-containing genomic sequences (Supplementary data, Table S1), indicating a specific regulatory region. We further assessed a possible regulatory functional effect of the SNP rs28470223 on the binding sites of transcription factors using the perfectos-ape tool (Vorontsov et al. 2015) based on the hocomoco v11 motif database (Kulakovskiy et al. 2018). The SNP rs28470223 alters binding of several transcription factors (Supplementary data, Table S2), predicting a functional effect of this SNP in modifying transcription factor binding affinities to rs28470223-containing genomic sequences. The P-value decreases more than 10-fold for the allele T compared with the allele C for the AP2D and KAISO transcription factors whereas it increases more than 10-fold for the BRAC and TCFP2 transcription factors (Supplementary data, Table S2). Finally, the SNP rs28470223 was found to be an expression quantitative trait locus (eQTL) (P = 3.06 10−6) in the Genotype-Tissue Expression database (Consortium 2013). Overall, the bioinformatics analysis supports a cis-regulatory effect of the SNP rs28470223. We next performed gene reporter assays to evaluate the functional effect of the SNP rs28470223 on the HS3ST3A1 promoter activity in K562 cells. As a result, the minor allele (rs28470223-T) significantly decreases the promoter activity by ~54% compared with the wild type allele (rs28470223-C) on the basis of six experiments including both alleles (P = 6 10−5) (Figure 1), confirming the regulatory function of rs28470223 on the promoter transcriptional activity of HS3ST3A1. Fig. 1. View largeDownload slide rs28470223 polymorphism alters HS3ST3A1 promoter activity. Six luciferase reporter gene assays with constructs containing the rs28470223-C (n = 6) or rs28470223-T (n = 6) HS3ST3A1 promoter in K562 cell line were performed. All constructs were co-transfected with pRL-SV40 to standardize transfection efficiency and luciferase activity for each sample was adjusted by the empty pGL3-Enhancer vector. Statistical analysis was performed using two-tailed Student’s t-test after controlling the normality of the data and the equality of their variance. Mean and standard error mean of relative luciferase activity are shown for each allele. Fig. 1. View largeDownload slide rs28470223 polymorphism alters HS3ST3A1 promoter activity. Six luciferase reporter gene assays with constructs containing the rs28470223-C (n = 6) or rs28470223-T (n = 6) HS3ST3A1 promoter in K562 cell line were performed. All constructs were co-transfected with pRL-SV40 to standardize transfection efficiency and luciferase activity for each sample was adjusted by the empty pGL3-Enhancer vector. Statistical analysis was performed using two-tailed Student’s t-test after controlling the normality of the data and the equality of their variance. Mean and standard error mean of relative luciferase activity are shown for each allele. A missense SNP is located within the catalytic domain of 3-OST-3 at proximity of the reducing end of the substrate The catalytic domain of human 3-OST-3B1 shares 99% sequence identity with that of 3-OST-3A1 for which the structure has been determined, arguing that this functionally important region is actively maintained by gene conversion between the 3-OST-3A and 3-OST-3B loci; each gene being duplicated in human (Shworak et al. 1999). The catalytic domain of 3-OST-3 displays an overall α/β motif typically found in sulfotransferases with a large open cleft running across the active site. Central to this structural motif are the phosphosulfate binding loop (P-loop like motif, Lys162-Arg166) of the high-energy sulfate donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) and a conserved glutamate acting as the catalytic base, a binding site well conserved in sulfotransferases (Liu et al. 2012). The acceptor site for sulfonation, referred as the acceptor sugar, lies at the center of the cleft and corresponds to the position of a GlcNS6S residue (T-2 in 3-OST-3/H-3 in 3-OST-1) (Figure 2) (Moon et al. 2004, 2012). Fig. 2. View largeDownload slide Mapping residues corresponding to missense SNP polymorphism on the crystal structure of 3-OST-3A1. (A) Overall view of the catalytic domain of the homologous 3-OST-3 dimer with each subunit colored in cyan and yellow. Mutated residues (orange) that severely affect the catalytic activity are mapped onto the solvent-accessible surface of human 3-OST-3 (accession code 1T8U) in complex with PAP (orange with magenta phosphate) and a tetrasaccharide (blue with green sulfate); those that correspond to the three 3-OST-3B1 SNPs are shown in red with the Ile196Val rs62056073 variant located on the back face. The Ile288Ala mutant, which is closely located to the Ser269Asn variant and less severely affects the catalytic activity, is shown in violet. (B) Close-up of boxed area, oriented as in panel A, of the active site of 3-OST-3 with bound PAP and tetrasaccharide. Fig. 2. View largeDownload slide Mapping residues corresponding to missense SNP polymorphism on the crystal structure of 3-OST-3A1. (A) Overall view of the catalytic domain of the homologous 3-OST-3 dimer with each subunit colored in cyan and yellow. Mutated residues (orange) that severely affect the catalytic activity are mapped onto the solvent-accessible surface of human 3-OST-3 (accession code 1T8U) in complex with PAP (orange with magenta phosphate) and a tetrasaccharide (blue with green sulfate); those that correspond to the three 3-OST-3B1 SNPs are shown in red with the Ile196Val rs62056073 variant located on the back face. The Ile288Ala mutant, which is closely located to the Ser269Asn variant and less severely affects the catalytic activity, is shown in violet. (B) Close-up of boxed area, oriented as in panel A, of the active site of 3-OST-3 with bound PAP and tetrasaccharide. Given that substrate specificity in HS O-sulfotransferases could be determined by residues distal to the catalytic site, we hypothesized that the three naturally occurring 3-OST-3 SNP isoforms would display differential 3-O-sulfotransferase activities compared with the wild type enzyme. To investigate potential functional effects of the coding SNPs on the 3-OST-3 catalytic activity, we mapped the three missense SNPs onto the crystal structure of 3-OST-3 in ternary complex with PAP and a tetrasaccharide (Moon et al. 2004). This structural comparison was extended to the crystal structure of 3-OST isoform 1 in ternary complex with PAP and a heptasaccharide (Moon et al. 2012). The three missense SNPs are located in loop regions at the surface and hardly alter enzyme functionality (Figure 2). Yet, the Ser269Asn SNP is located at the tip of helix α8 and faces the entry of the large substrate binding at site T-4/H-5 on the reducing end at proximity of the GlcNS6S N-sulfate group (tetrasaccharide complex in 3-OST-3) or 6-O-sulfate group (heptasaccharide complex in 3-OST-1). Sequence analysis of a subset of the 3-O-sulfotransferase family members reveals that Ser269 is conserved across mammalian 3-OST-3 orthologs but variability is tolerated at this position in other 3-OST-3 isoforms (Asn in 3-OST-2 and Lys in 3-OST-1/5). The Ser269Asn SNP variant exhibits a slightly higher catalytic activity We expressed and purified human wt 3-OST-3B1 and the Ser269Asn variant using a mammalian cell expression system and anti-FLAG affinity purification tag. Expression of wt 3-OST-3B1 and the Ser269Asn variant clearly shows a band at 37 kDa by western blot analysis corresponding to the recombinant proteins. We used an HS-derived octasaccharide substrate (HS8) as a mimic of a natural HS substrate to identify with a high pressure liquid chromatography (HPLC)-based assay any differences in enzyme activities between the wild type and the Ser269Asn variant. HS8 was prepared by depolymerization of HS with heparinase III, an enzyme that specifically cleaves GlcNAc-containing disaccharide units, thereby releasing intact NS-domain-like oligosaccharides. Digestion products were purified using combined size-exclusion and anion-exchange chromatography to yield size and charge homogenous oligosaccharide species (Pye et al. 1998). Amongst these, HS8 was selected for the 3-OST enzymatic assay as this oligosaccharide displays an average sulfation content suitable for 3-OST-3B1 activity (Supplementary data, Figure S1). HS8 was incubated with wt 3-OST-3B1 or the Ser269Asn variant and reaction was monitored by measuring the disaccharide composition using a dedicated HPLC-based assay (see Materials and methods). HPLC analysis of disaccharides present in untreated HS8 yielded four major peaks, enabling determination of the disaccharide composition after integration and normalization of the peak areas (Supplementary data, Table S3). In contrast, a similar disaccharide analysis of 3-OST-3B1-treated HS8 showed the appearance and time-dependent increase of an unknown, late-eluting saccharide species (Figure 3). This species most likely corresponds to a heparinase-resistant 3-O-sulfated tetrasaccharide, as previously reported (Yamada et al. 1993). Concomitantly, the 3-OST-3B1 activity decreased the ΔHexA,2S-GlcNS disaccharide content while the pattern of other HS8 disaccharides (i.e. ΔHexA,GlcNAc, ΔHexA,GlcNS and ΔHexA,2S-GlcNS6S) remained unchanged (Figure 3 and Supplementary data, Figure S2). These data clearly indicate that the 3-OST-3B1-caltalyzed 3-O-sulfation event on HS8 is non-random and may preferentially target IdoA2S-GlcNS units, in agreement with previous studies and on the fact that the 3-OST-3 modification must precede the 6-O-sulfation step (Liu, Shworak, et al. 1999; Thacker et al. 2014; Wang et al. 2017). Instead, the 3-OST-1 modification occurs only after 6-O-sulfation to generate the -GlcA,GlcNS3S6S- disaccharide unit (Wang et al. 2017). Fig. 3. View largeDownload slide Analysis of 3-OST-3B1-catalyed 3-O-sulfation of HS8 oligosaccharide. (A) RPIP-HPLC-elution profiles of untreated (gray trace) and 3-OST-3B1-treated (black trace) HS8 oligosaccharides. The peak (marked *) eluted at the highest NaCl concentration corresponds to a new 3-OST-3B1-catalyzed 3-O-sulfated species. (B) Time course of 3-O-sulfation of HS8 by wt 3-OST-3B1 (open circles) and the Ser269Asn mutant (closed circles) showing the evolution of the disaccharide content in ΔHexA,GlcNAc (left), unknown species (peak *, middle) and ΔHexA,2S-GlcNS (right). ΔHexUA-GlcNAc (left) and ΔHexUA,2S-GlcNS (right) contents have been normalized and expressed as a percentage of all disaccharides detected. The amount of the * species (middle) has been normalized using arbitrary units and expressed as a percentage of the total fluorescence signal detected. Fig. 3. View largeDownload slide Analysis of 3-OST-3B1-catalyed 3-O-sulfation of HS8 oligosaccharide. (A) RPIP-HPLC-elution profiles of untreated (gray trace) and 3-OST-3B1-treated (black trace) HS8 oligosaccharides. The peak (marked *) eluted at the highest NaCl concentration corresponds to a new 3-OST-3B1-catalyzed 3-O-sulfated species. (B) Time course of 3-O-sulfation of HS8 by wt 3-OST-3B1 (open circles) and the Ser269Asn mutant (closed circles) showing the evolution of the disaccharide content in ΔHexA,GlcNAc (left), unknown species (peak *, middle) and ΔHexA,2S-GlcNS (right). ΔHexUA-GlcNAc (left) and ΔHexUA,2S-GlcNS (right) contents have been normalized and expressed as a percentage of all disaccharides detected. The amount of the * species (middle) has been normalized using arbitrary units and expressed as a percentage of the total fluorescence signal detected. A kinetic analysis of the Ser269Asn variant activity compared with wt 3-OST-3B1 revealed similar elution profiles, with a slightly faster increase of the peak * area associated to a concomitant faster decrease of the ΔHexA,2S-GlcNS disaccharide content, as monitored by a time course treatment (Figure 3). This result points to a slightly higher catalytic activity for the Ser269Asn variant compared with the wt enzyme. In fact, structural analysis suggests that a bulkier Asn in the 3-OST-3B1 variant could provide additional polar interactions with the N- or 6-O-sulfate group of GlcNS6S at site T-4 near the reducing end of the substrate compared with a shorter Ser residue in wt 3-OST3. Site-directed mutagenesis of the adjacent Trp268 residue in 3-OST-3, which contributes to a sulfate-binding pocket near the reducing end of the substrate, resulted in a 41-fold reduction in catalytic efficiency, arguing for a functional role of the contiguous Ser269 in substrate binding (Moon et al. 2012). In addition to Ser269, other residues remote from the active site but located at the reducing end of the substrate, including Thr256 in 3-OST-3 and Arg268 in 3-OST-1, were shown to play important roles in substrate binding and specificity between 3-OST isoforms (Moon et al. 2012). Overall, this first genetic and biochemical analyses of two genetic variants in the HS3ST3A1/B1 genes associated with P. falciparum parasitaemia offer first insights into the modulation of 3-OST-3 gene transcription and catalytic activity by SNPs. Whether the SNPs affected HSPG level and structure in the population living in an endemic area in Burkina Faso is unknown and merits further investigation. Materials and methods In silico analysis of the SNP regulatory effect We explored ChIP-seq results by using the ReMap atlas covering 485 transcription factors to identify transcription factors that bind DNA sequence containing the studied SNPs (Chèneby et al. 2018). To this aim, we used the ReMap tool to intersect the genomic coordinates of SNPs with those of the ChIP-seq peaks. Furthermore, we used the PERFECTOS-APE software to predict the effect of the SNPs on the binding of transcription factors (Vorontsov et al. 2015). Briefly, the extracted SNPs sequences and their alleles together with their flanking regions were scanned against a recent collection of motifs, which contains binding models of 680 human transcription factors (Kulakovskiy et al. 2018). The site score was obtained from position-specific scoring matrices on sequences containing each allele, allowing us to assess the P-value for each allele and therefore the P-value ratio. The candidate transcription factors were retained on the basis of the best P-value obtained for one of the allele; it should be lower than 10−3. The calculated P-value ratio reflects the predicted effect of the SNP on the binding of the transcription factor on the SNP-containing DNA sequence. Transient transfection and dual-luciferase reporter assay K562 cells (ATCC CLL-243) were grown in Gibco RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS). A 1200 bp DNA fragment upstream the HS3ST3A1 translation start site (chromosome 17:13504472-13505672 according to the hg38 assembly) was cloned by gene synthesis (GeneCust Custom Services, Luxembourg). This fragment was cloned into the MluI-XhoI sites of the pGL3-basic vector (Promega, Madison, WI, USA), which contains the firefly luciferase coding sequence. Initially, the pGL3 construct contained the rs28470223 T-allele which represents the minor allele in Africa, whereas the rs28470223-C is the major allele in Africa and is considered as the wild type allele. To generate the pGL3 construct containing the rs28470223-C allele, site-directed mutagenesis was performed with the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA) using primers (5′-AGGTTCTCTCGGCCAAGGAGC-3′, 3′-GAACTCGGGCGGAGAGAA-5′) designed by NEBaseChanger tool and provided by the supplier, and an annealing temperature of 63°C. K562 transfection was performed with the Neon™ Transfection System (Invitrogen) following manufacturer’s instructions. For each of the six tests performed, 106 cells were co-transfected with 1 μg of the empty pGL3-basic control vector, or with positive pGL3-promoter control vector, or with 1 μg of pGL3-basic vector containing the rs28470223-T (p-T) or rs28470223-C (p-C) HS3ST3A1 promoter sequence together with 200 ng of the pRL-SV40 plasmid encoding Renilla luciferase (Promega, Madison, WI, USA), which was used as a transfection efficiency control. Transfected cells were maintained at 37°C in 5% CO2 during 24 h. Firefly and Renilla luciferase activities were monitored using a TriStar LB 941 Multimode Microplate Reader (Berthold technologies, Thermo Fisher Scientific, Waltham, MA, USA) by analyzing 20 μl of cell lysate according to the manufacturer’s instructions provided in the Dual-Luciferase Kit (Promega). The firefly luciferase activity of each sample was normalized by the Renilla luciferase activity and adjusted by the pGL3-basic mean activity. Statistical analysis To assess the effect of SNPs on the promoter activity, Student’s t-test was used after checking the normality of the distributions by using the Shapiro–Wilk method and their variance equality by using a Fisher test. All analyses were performed by using either R or SPSS software (SPSS, Boulogne, France). Only significant terms at the 5% level were retained. Expression and purification of human 3-OST-3B1 and the Ser269Asn variant Following unsuccessful attempts to express a soluble form of the human 3-OST-3B1 catalytic domain (Ile125-Asp390) in Escherichia coli, either as a 6xHis- or thioredoxine-tagged form, a cDNA fragment encoding the catalytic domain of wt 3-OST-3B1 was cloned into the pYD7 vector suitable for expression in human embryonic kidney HEK293 EBNA cell line. The recombinant pYD7-3-OST-3B1 vector consists of a Kozak consensus sequence, a SEAP peptide signal and a Flag tag upstream of the catalytic domain for 3-OST-3B1 detection and purification. The pYD7 plasmid carrying the 3-OST-3B1 Ser269Asn variant (rs61729712) was amplified by polymerase chain reaction (PCR) from the wild type construct using the forward 5′-TCGACACGTCGTGGAACGCCATCCAGATCGG-3′ and reverse 5′-CCGATCTGGATGGCGTTCCACGACGTGTCGA-3′ oligonucleotides. The PCR product was cloned into the linearized PYD7 plasmid DNA and the sequence was verified by sequencing. Plasmid DNA (2 μg) was transfected with 4 μg of linear polyethylenimine (Polysciences) into HEK293 EBNA cells in a 6-well culture plate. Transfected cells cultured in Dulbecco's modified Eagle's medium (DMEM) 10% FBS were selected with blasticidin (5 μg/ml) for stable cell line establishment. Higher expression and secretion levels of the protein produced by the cell lines were achieved in DMEM 2% FBS, 1.25 mM sodium valproate (Sigma-Aldrich) and 0.5% Tryptone N1 (Organotechnie). Protein production was performed in multilayer culture flasks at 32°C instead 37°C to reduce protein aggregation. Protein secretion was analyzed by western blot using an anti-FLAG tag antibody. Before purification, culture medium containing secreted protein was harvested after 7 days of culture and dialyzed overnight at 4°C against 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 8.0 and 100 mM NaCl. FLAG-tagged wt 3-OST-3B1 was purified by anti-FLAG M2 affinity (Sigma-Aldrich) and eluted in a buffer containing 20 mM HEPES pH 8.0, 100 mM NaCl and 100 μg/ml FLAG tag peptide (Sigma-Aldrich). Fractions containing 3-OST-3B1 were pooled and further purified by size-exclusion chromatography on a Superdex 200 26/60 column (GE-Healthcare) equilibrated with 20 mM Tris pH 8.0 and 200 mM NaCl. The Ser269Asn variant was produced and purified as for wt 3-OST-3B1. Protein purity and integrity of wt 3-OST-3B1 and the Ser269Asn variant were analyzed by SDS-PAGE electrophoresis and matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry, and concentrated by ultrafiltration to 1 mg/ml in 20 mM Tris-HCl pH 8.0, 100 mM NaCl and 1 mM PAP (Sigma-Aldrich). Preparation of HS-derived octasaccharides Porcine mucosal HS (Celsius) was extensively digested with 25 mU/ml of heparinase III (Grampian Enzyme) in 5 mM Tris-HCl, 2 mM CaCl2, 0.1 mg/ml BSA, 50 mM NaCl, pH 7.5 for 72 h at 30°C. The resulting mixture was resolved onto a 1500 mm × 44 mm Biogel P10 column (Bio-Rad) in 200 mM NaCl at 1 ml/min, leading to a graded series of size-uniform oligosaccharides from disaccharide to octadecasaccharide. Samples were dialyzed against distilled water, freeze-dried and quantified. The octasaccharides were further purified by strong-anion-exchange HPLC, on a 9 × 250 mm preparative ProPac PA1 column (Dionex). After equilibration in mobile phase (distilled water adjusted to pH 3.5 with HCl) at 1 ml/min, samples (7.5 mg) were injected and eluted with a gradient of NaCl (0–0.5 M over 10 min, then 0.5–1 M over 80 min) in the same mobile phase. The eluate was monitored online for UV absorbance at 232 nm, and 23 different fractions were collected, dialyzed against distilled water, freeze-dried, quantified and analyzed as described below. Fraction HS8 was used as a substrate to monitor the 3-OST-3B1 catalytic activity. Time course of 3-O-sulfation of the HS8 oligosaccharide by 3-OST-3B1 Lyophilized HS8 was resuspended in 50 mM MES pH 7.0, 5 mM MgCl2, 200 μM PAPS and incubated with 1 μg of 3-OST-3B1 (wild type or the Ser269Asn variant) for 1 h at 37°C. For time course digestion, aliquots were taken off at 0, 1, 3, 6 and 24 h time points in triplicate. Samples were then boiled for 5 min to inactive the enzyme and then stored at −20°C. Disaccharide analysis Disaccharide analysis of the HS8 samples was performed as previously described (Henriet et al. 2017). Briefly, samples in 100 mM sodium acetate, 0.5 mM calcium acetate, pH 7.1 were digested into disaccharides by incubation with a cocktail of heparinase I, II and III (10 mU each) overnight at 37°C. These experimental conditions are not suitable for the 3-O-sulfation specific peeling reaction. Disaccharide composition was determined by RPIP-HPLC, by injection on a Luna 5 μm C18 reversed phase column (4.6 × 300 mm, Phenomenex, Le Pecq, France) equilibrated at 0.5 ml/min in 1.2 mM tetra-N-butylammonium hydrogen sulfate (TBA) in 8.5% acetonitrile. Disaccharides were then resolved using a multi-step NaCl gradient (0–30 mM in 1 min, 30–90 mM in 39 min, 90–228 mM in 2 min, 228 mM for 4 min, 228–300 mM in 2 min, 300 mM for 4 min). Online post-column disaccharide derivatization was achieved by addition of 2-cyanoacetamide (0.25%) in NaOH (0.5%) at a flow rate of 0.16 ml/min, followed by fluorescence detection (excitation 346 nm, emission 410 nm). Fluorescence signal was normalized with HS disaccharide standards (Iduron, Alderley Edge, UK) or using arbitrary units for the unknown (peak *) saccharide species in absence of standards. Direct comparison of these normalized disaccharide contents is possible, unlike that of the unknown (peak *) species. Structural analysis A structural analysis was performed to map the three 3-OST-3B1 missense SNPs on the crystal structure of 3-OST-3 bound to PAP and a tetrasaccharide substrate (accession code 1T8U) or 3-OST-1 bound to PAP and heptasaccharide substrate (3UAN). The root-mean-square deviation value between the two structures is 0.95 Å for 244Cα atoms (44% sequence identity). Supplementary data Supplementary data is available at GLYCOBIOLOGY online. Funding This work was supported in part by the CNRS, INSERM and Aix-Marseille University to P.R./Y.B., the GDR GAG (GDR 3739) to R.R.V./H.L-J., the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-05-01 to Y.B./H.L-J., the LabEx GRAL (ANR-10-LABX-49-01) and the “Investissements d’avenir” program Glyco@Alps (ANR-15-IDEX-02) to H.L-J./R.R.V. Acknowledgements TNN was supported by a PhD fellowship from the Vietnamese government. We thank Melanie Daligault (TAGC, Marseille) for helpful assistance with bioinformatics analyses, Ahmad Ali-Ahmad and Pascale Marchot (AFMB, Marseille) for helpful assistance with biochemical analyses. Conflict of interest statement None declared. 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Structural studies on the bacterial lyase-resistant tetrasaccharides derived from the antithrombin III-binding site of porcine intestinal heparin. J Biol Chem . 268: 4780– 4787, doi:8444855. Google Scholar PubMed  © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Glycobiology Oxford University Press

Genetic and enzymatic characterization of 3-O-sulfotransferase SNPs associated with Plasmodium falciparum parasitaemia

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Abstract

Abstract The HS3ST3A1/B1 genes encode two homologous 3-O-sulfotransferases involved in the late modification step during heparan sulfate (HS) biosynthesis. In addition to the single nucleotide polymorphisms (SNPs) rs28470223 (C > T) in the promoter region of both HS3ST3A1 and rs62636623 (Gly/Arg) in the stem region of HS3ST3B1, three missense mutations (rs62056073, rs61729712 and rs9906590) located within the catalytic sulfotransferase domain of 3-OST-B1 are linked and associated to Plasmodium falciparum parasitaemia. To ascertain the functional effects of these SNP associations, we investigated the regulatory effect of rs28470223 and characterized the enzymatic activity of the missense SNP rs61729712 (Ser279Asn) localized at proximity of the substrate binding cleft. The SNP rs28470223 results in decreased promoter activity of HS3ST3A1 in K562 cells, suggesting a reduced in vivo transcription activity of the target gene. A comparative kinetic analysis of wt HS3ST3B1 and the Ser269Asn variant (rs61729712) using a HS-derived oligosaccharide substrate reveals a slightly higher catalytic activity for the SNP variant. These genetic and enzymatic studies suggest that genetic variations in enzymes responsible of HS 3-O-sulfation can modulate their promoter and enzymatic activities and may influence P. falciparum parasitaemia. enzymatic activity, genetic association, heparan sulfate biosynthesis, 3-O-sulfotransferase Introduction Heparan sulfate (HS) is a family of highly N- and O-sulfated glycosaminoglycans that is commonly found on the mammalian cell surface and in the extracellular matrix. HS is attached to a variety of different core proteins defined as HS proteoglycans (HSPGs) and participates in a wide range of physiological and pathophysiological functions, including embryonic development, inflammatory responses, blood coagulation and assisting viral/bacterial infections (Bishop et al. 2007). HS consists of a disaccharide repeating unit with D-glucuronic acid (GlcA) or L-iduronic acid (IdoA) and glucosamine, each of which is capable of carrying sulfate groups and contributes to dictate HS function (Bishop et al. 2007). The HS maturation process involves a series of biosynthetic enzymes, including bifunctional enzymes (NSDT, N-deacetylase/N-sulfotransferase), a critical step for formation of N-sulfated domains, a C5-epimerase and various O-sulfotransferases (Fu et al. 2016). The three classes of O-sulfotransferases transfer sulfate groups at various positions, including C2 of IdoA, C6 and C3 of the GlcN units, leading to clusters of highly sulfated regions (NS domain) separated by long stretches of unsulfated GlcNAc-containing disaccharides (NA domain). The sulfate-rich regions are likely to generate highly flexible regions because of their high content in the conformationally versatile IdoA and IdoA2S residues (Turnbull and Gallagher 1991). In addition, tissue specific expression of different enzyme isoforms fine-tunes synthesis of specific HS structures, which are predominantly determined by a controlled positioning of N-, 2-, 6- and 3-O-sulfate groups along HS chains, to confer an important regulatory role in various biological processes (Esko and Selleck 2002). The 3-O-sulfation of GlcN unit is a relatively rare modification, present in only a limited number of HS polymers or absent entirely, and occur at a late step during the biosynthetic pathway (Thacker et al. 2014). In this context, the natural abundance of HS 3-O-sulfation may be re-evaluated given the occurrence of a peeling reaction that specifically degrades 3-O-sulfated GlcN unit at the reducing ends of enzymatically depolymerized HS/heparin chains (Huang et al. 2015). Conversely, the HS 3-O-sulfotransferases (3-OST-3 that are encoded by HS3ST3 genes) represent the largest gene family among all HS sulfotransferases and vertebrates generally possess seven isozymes divided into two subgroups according to sequence homology of their sulfotransferase domain (Liu and Pedersen 2007). Based on the large diversity of 3-OST enzymes and the fact that they probably act at a late stage, one might assume that they show selectivity for the sulfation pattern in their target sites. One group (3-OST-2,-3A,-3B,-4 and -6) is often referred to as “gD-type” HS3STs, because members of the subfamily can generate binding sites for glycoprotein gD of type I herpes simplex virus (HSV-1). Members of the second group (3-OST-1, -5) have in common the capacity to generate a binding site for antithrombin and are thus designed “AT-type” HS3STs. Hence, 3-OST-1 preferentially modifies GlcN sites in which GlcA at position +1 is devoid of 2-O-sulfate groups (Mochizuki et al. 2008). In contrast, 3-OST-2, -3, -4 and -6 (gD-type) preferentially modify GlcN sites in which position -1 is IdoA2S (Meissen et al. 2009). Unlike 3-OST-1, the 3-OST-2, -3, -3A and -3B enzymes are predicted to have type II integral membrane architecture (Shworak et al. 1999). Both mouse and human forms of 3-OST-3B and a human form of 3-OST-3A can modify the HS of HSV-1-resistant cells at specific sites to generate cells susceptible to HSV-1 infection (Shukla et al. 1999). In addition to their functional implications in various cell events, HS proteoglycans can play an important role in infectious diseases, such as malaria caused by the parasite Plasmodium, involving the HS chains of both the mammalian host and the vector. Several studies suggest that the outcome of malaria infection may be influenced by differences in HS composition, owing to genetic variations within genes encoding the biosynthesis enzymes (Coppi et al. 2007; Sinnis et al. 2007; Armistead et al. 2011). The genes encoding the 3-O-sulfotransferases are located in different chromosomal regions except for HS3ST3A1 and HS3ST3B1 which encode the 3-OST-3A and 3-OST-3B sulfotransferases, respectively, and target the same disaccharide (Liu, Shriver, et al. 1999). In fact, those two genes are located within chromosome 17p12 that showed a genome-wide significant linkage with Plasmodium falciparum parasitaemia in a population living in Burkina Faso (Brisebarre et al. 2014). Moreover, genetic variations within HS3ST3A1 and HS3ST3B1 are linked to P. falciparum parasitaemia in humans, as found in a population living in an endemic area in Burkina Faso (Atkinson et al. 2012). Alteration of HS 3-O-sulfation may affect (i) the binding of P. falciparum antigen on host cells and/or (ii) the pro-inflammatory response. To date, several single nucleotide polymorphisms (SNPs) have been identified in the promoter region of both HS3ST3A1/B1, and one of them (rs28470223, C/T), which is located within the promoter of HS3ST3A1, was linked and associated with P. falciparum parasitaemia. Moreover, four missense SNPs (rs62636623 G > C Gly83Arg, rs62056073 A > G Ile196Val, rs61729712 G > A Ser269Asn and rs9906590 G > A Glu363Lys) are located within HS3ST3B1, of which the later three SNPs are located within the sulfotransferase domain. These missense SNPs were genetically linked to parasitaemia and were associated with parasitaemia in combination with other SNPs, suggesting that they may alter 3-OST-3 functionality. While epidemiological correlation studies relate naturally occurring variations in 3-O-sulfotransferases to the susceptibility for infectious diseases in these populations, no functional data are yet available for most of these genetic variants. To ascertain the functional role of the SNPs localized either in the promotor region or within the sulfotransferase domain of the HS 3-OST-3 modification enzyme, we investigated the effect of the SNP rs28470223 on the expression level of the HS3ST3A1 gene and the effect of the Ser269Asn (rs61729712) variant on the enzymatic activity of human 3-OST-3B1. Bioinformatics analyses reveal that seven SNPs in HS3ST3A1/B1 show a predicted deleterious effect, confirming that genetic variations within the genes encoding 3-O-sulfotransferases may affect the susceptibility to infectious diseases, such as malaria. The SNP rs28470223 is associated with a significantly altered promoter activity that may affect both the transcriptional activity and P. falciparum parasitaemia in humans. Complementary enzyme kinetic analyses of wt human 3-OST-3 and the Ser269Asn (rs61729712) variant, which is located at the entry of the substrate binding cleft based on the crystal structure of the homologous 3-OST-3A1 isoform, reveal a slightly higher catalytic activity compared with the wt enzyme. Results and discussion Bioinformatics analysis of the phenotypic effect of HS3ST3A1 and HS3ST3B1 genetic variants All SNPs previously identified in the chromosomal region containing HS3ST3A1 and HS3ST3B1 from a population living in an endemic area in Burkina Faso (Atkinson et al. 2012) were investigated using the bioinformatics PredictSNP2 tool, which integrates binary predictions and uniform confidence values computed by six different tools. The comparative analysis, which provides a consensus score based on the five best-performing tools, reveals that seven SNPs show a predicted deleterious effect, including three (rs62056073 A > G Ile196Val, rs61729712 G > A Ser269Asn and rs9906590 G > A Glu363Lys) out of the four missense variants (Table I). In contrast, rs28470223 C > T could not be predicted for deleterious effects using the PredictSNP2 tool in contrast to the alternative FunSeq2 and GWAVA predicting tools. Table I. Predicted effect for HS3ST3A1 and HS3ST3B1 variants SNP_ID  GRCh37 location (bp)  Variant  Gene location  Type of change  PredictSNP2  CADD  DANN  FATHMM  FunSeq2  GWAVA  rs62057033  13399616  A > T  HS3ST3A1  Exon 2 - Synonymous  96% Neutral  73% Neutral  90% Neutral  77% Neutral  93% Neutral  56% Neutral  rs61732181  13399928  C > T  HS3ST3A1  Exon 2 - Synonymous  88% Neutral  58% Deleterious  90% Neutral  93% Deleterious  93% Neutral  56% Neutral  rs61744056  13400057  T > C  HS3ST3A1  Exon 2 - Synonymous  91% Neutral  94% Neutral  97% Neutral  90% Neutral  93% Neutral  62% Deleterious  rs8080565  13400153  G > A  HS3ST3A1  Intron  77% Neutral  86% Neutral  54% Deleterious  85% Neutral  80% Neutral  58% Neutral  rs3744337  13504665  A > G  HS3ST3A1  5′UTR  74% Neutral  67% Deleterious  62% Neutral  93% Neutral  83% Neutral  64% Deleterious  rs3744335  13504884  T > G  HS3ST3A1  5′UTR  88% Neutral  86% Neutral  80% Neutral  93% Neutral    53% Neutral  rs28470223  13505023  A > G  HS3ST3A1  5′UTR  88% Neutral  76% Neutral  86% Neutral  91% Neutral  67% Deleterious  64% Deleterious  rs78863672  13505237  C > A  HS3ST3A1  5′UTR  91% Deleterious  86% Deleterious  62% Neutral  86% Deleterious  67% Deleterious  84% Deleterious  rs2072243  14204380  C > T  HS3ST3B1  5′UTR  97% Deleterious  76% Deleterious  89% Deleterious    68% Deleterious  80% Deleterious  rs2072242  14204410  T > C  HS3ST3B1  5′UTR  97% Deleterious  98% Deleterious  89% Deleterious  88% Deleterious  62% Deleterious  78% Deleterious  rs115229628  14204423  G > A  HS3ST3B1  5′UTR  97% Deleterious  98% Deleterious  100% Deleterious  91% Deleterious  67% Deleterious  76% Deleterious  rs62636623  14205082  G > C  HS3ST3B1  Exon 1 - Missense Gly > Arg  89% Neutral  94% Neutral  90% Neutral  77% Neutral  84% Neutral    rs62636622  14205168  G > A  HS3ST3B1  Exon 1 - Synonymous  96% Neutral  92% Neutral  90% Neutral  87% Neutral  93% Neutral  54% Neutral  rs62056073  14248376  A > G  HS3ST3B1  Exon 2 - Missense Ile > Val  87% Deleterious  58% Neutral  60% Deleterious  59% Deleterious  62% Neutral  51% Deleterious  rs9906855  14248423  T > C  HS3ST3B1  Exon 2 - Synonymous  96% Neutral  94% Neutral  97% Neutral  90% Neutral  93% Neutral  62% Deleterious  rs61729712  14248596  G > A  HS3ST3B1  Exon 2 - Missense Ser>Asn  87% Deleterious  53% Deleterious  60% Deleterious  82% Deleterious  62% Neutral  51% Deleterious  rs55688668  14248702  G > A  HS3ST3B1  Exon 2 - Synonymous  93% Neutral  83% Neutral  90% Neutral  57% Deleterious  93% Neutral  56% Neutral  rs9906590  14248877  G > A  HS3ST3B1  Exon 2 - Missense Glu > Lys  87% Deleterious  53% Deleterious  60% Deleterious  65% Deleterious  62% Neutral  50% Deleterious  rs3785655  14249167  C > T  HS3ST3B1  3′UTR  88% Neutral  86% Neutral  68% Neutral  75% Neutral  83% Neutral  53% Neutral  rs73979332  14249433  C > T  HS3ST3B1  3′UTR  88% Neutral  80% Neutral  62% Neutral  89% Neutral  81% Neutral  71% Neutral  SNP_ID  GRCh37 location (bp)  Variant  Gene location  Type of change  PredictSNP2  CADD  DANN  FATHMM  FunSeq2  GWAVA  rs62057033  13399616  A > T  HS3ST3A1  Exon 2 - Synonymous  96% Neutral  73% Neutral  90% Neutral  77% Neutral  93% Neutral  56% Neutral  rs61732181  13399928  C > T  HS3ST3A1  Exon 2 - Synonymous  88% Neutral  58% Deleterious  90% Neutral  93% Deleterious  93% Neutral  56% Neutral  rs61744056  13400057  T > C  HS3ST3A1  Exon 2 - Synonymous  91% Neutral  94% Neutral  97% Neutral  90% Neutral  93% Neutral  62% Deleterious  rs8080565  13400153  G > A  HS3ST3A1  Intron  77% Neutral  86% Neutral  54% Deleterious  85% Neutral  80% Neutral  58% Neutral  rs3744337  13504665  A > G  HS3ST3A1  5′UTR  74% Neutral  67% Deleterious  62% Neutral  93% Neutral  83% Neutral  64% Deleterious  rs3744335  13504884  T > G  HS3ST3A1  5′UTR  88% Neutral  86% Neutral  80% Neutral  93% Neutral    53% Neutral  rs28470223  13505023  A > G  HS3ST3A1  5′UTR  88% Neutral  76% Neutral  86% Neutral  91% Neutral  67% Deleterious  64% Deleterious  rs78863672  13505237  C > A  HS3ST3A1  5′UTR  91% Deleterious  86% Deleterious  62% Neutral  86% Deleterious  67% Deleterious  84% Deleterious  rs2072243  14204380  C > T  HS3ST3B1  5′UTR  97% Deleterious  76% Deleterious  89% Deleterious    68% Deleterious  80% Deleterious  rs2072242  14204410  T > C  HS3ST3B1  5′UTR  97% Deleterious  98% Deleterious  89% Deleterious  88% Deleterious  62% Deleterious  78% Deleterious  rs115229628  14204423  G > A  HS3ST3B1  5′UTR  97% Deleterious  98% Deleterious  100% Deleterious  91% Deleterious  67% Deleterious  76% Deleterious  rs62636623  14205082  G > C  HS3ST3B1  Exon 1 - Missense Gly > Arg  89% Neutral  94% Neutral  90% Neutral  77% Neutral  84% Neutral    rs62636622  14205168  G > A  HS3ST3B1  Exon 1 - Synonymous  96% Neutral  92% Neutral  90% Neutral  87% Neutral  93% Neutral  54% Neutral  rs62056073  14248376  A > G  HS3ST3B1  Exon 2 - Missense Ile > Val  87% Deleterious  58% Neutral  60% Deleterious  59% Deleterious  62% Neutral  51% Deleterious  rs9906855  14248423  T > C  HS3ST3B1  Exon 2 - Synonymous  96% Neutral  94% Neutral  97% Neutral  90% Neutral  93% Neutral  62% Deleterious  rs61729712  14248596  G > A  HS3ST3B1  Exon 2 - Missense Ser>Asn  87% Deleterious  53% Deleterious  60% Deleterious  82% Deleterious  62% Neutral  51% Deleterious  rs55688668  14248702  G > A  HS3ST3B1  Exon 2 - Synonymous  93% Neutral  83% Neutral  90% Neutral  57% Deleterious  93% Neutral  56% Neutral  rs9906590  14248877  G > A  HS3ST3B1  Exon 2 - Missense Glu > Lys  87% Deleterious  53% Deleterious  60% Deleterious  65% Deleterious  62% Neutral  50% Deleterious  rs3785655  14249167  C > T  HS3ST3B1  3′UTR  88% Neutral  86% Neutral  68% Neutral  75% Neutral  83% Neutral  53% Neutral  rs73979332  14249433  C > T  HS3ST3B1  3′UTR  88% Neutral  80% Neutral  62% Neutral  89% Neutral  81% Neutral  71% Neutral  Table I. Predicted effect for HS3ST3A1 and HS3ST3B1 variants SNP_ID  GRCh37 location (bp)  Variant  Gene location  Type of change  PredictSNP2  CADD  DANN  FATHMM  FunSeq2  GWAVA  rs62057033  13399616  A > T  HS3ST3A1  Exon 2 - Synonymous  96% Neutral  73% Neutral  90% Neutral  77% Neutral  93% Neutral  56% Neutral  rs61732181  13399928  C > T  HS3ST3A1  Exon 2 - Synonymous  88% Neutral  58% Deleterious  90% Neutral  93% Deleterious  93% Neutral  56% Neutral  rs61744056  13400057  T > C  HS3ST3A1  Exon 2 - Synonymous  91% Neutral  94% Neutral  97% Neutral  90% Neutral  93% Neutral  62% Deleterious  rs8080565  13400153  G > A  HS3ST3A1  Intron  77% Neutral  86% Neutral  54% Deleterious  85% Neutral  80% Neutral  58% Neutral  rs3744337  13504665  A > G  HS3ST3A1  5′UTR  74% Neutral  67% Deleterious  62% Neutral  93% Neutral  83% Neutral  64% Deleterious  rs3744335  13504884  T > G  HS3ST3A1  5′UTR  88% Neutral  86% Neutral  80% Neutral  93% Neutral    53% Neutral  rs28470223  13505023  A > G  HS3ST3A1  5′UTR  88% Neutral  76% Neutral  86% Neutral  91% Neutral  67% Deleterious  64% Deleterious  rs78863672  13505237  C > A  HS3ST3A1  5′UTR  91% Deleterious  86% Deleterious  62% Neutral  86% Deleterious  67% Deleterious  84% Deleterious  rs2072243  14204380  C > T  HS3ST3B1  5′UTR  97% Deleterious  76% Deleterious  89% Deleterious    68% Deleterious  80% Deleterious  rs2072242  14204410  T > C  HS3ST3B1  5′UTR  97% Deleterious  98% Deleterious  89% Deleterious  88% Deleterious  62% Deleterious  78% Deleterious  rs115229628  14204423  G > A  HS3ST3B1  5′UTR  97% Deleterious  98% Deleterious  100% Deleterious  91% Deleterious  67% Deleterious  76% Deleterious  rs62636623  14205082  G > C  HS3ST3B1  Exon 1 - Missense Gly > Arg  89% Neutral  94% Neutral  90% Neutral  77% Neutral  84% Neutral    rs62636622  14205168  G > A  HS3ST3B1  Exon 1 - Synonymous  96% Neutral  92% Neutral  90% Neutral  87% Neutral  93% Neutral  54% Neutral  rs62056073  14248376  A > G  HS3ST3B1  Exon 2 - Missense Ile > Val  87% Deleterious  58% Neutral  60% Deleterious  59% Deleterious  62% Neutral  51% Deleterious  rs9906855  14248423  T > C  HS3ST3B1  Exon 2 - Synonymous  96% Neutral  94% Neutral  97% Neutral  90% Neutral  93% Neutral  62% Deleterious  rs61729712  14248596  G > A  HS3ST3B1  Exon 2 - Missense Ser>Asn  87% Deleterious  53% Deleterious  60% Deleterious  82% Deleterious  62% Neutral  51% Deleterious  rs55688668  14248702  G > A  HS3ST3B1  Exon 2 - Synonymous  93% Neutral  83% Neutral  90% Neutral  57% Deleterious  93% Neutral  56% Neutral  rs9906590  14248877  G > A  HS3ST3B1  Exon 2 - Missense Glu > Lys  87% Deleterious  53% Deleterious  60% Deleterious  65% Deleterious  62% Neutral  50% Deleterious  rs3785655  14249167  C > T  HS3ST3B1  3′UTR  88% Neutral  86% Neutral  68% Neutral  75% Neutral  83% Neutral  53% Neutral  rs73979332  14249433  C > T  HS3ST3B1  3′UTR  88% Neutral  80% Neutral  62% Neutral  89% Neutral  81% Neutral  71% Neutral  SNP_ID  GRCh37 location (bp)  Variant  Gene location  Type of change  PredictSNP2  CADD  DANN  FATHMM  FunSeq2  GWAVA  rs62057033  13399616  A > T  HS3ST3A1  Exon 2 - Synonymous  96% Neutral  73% Neutral  90% Neutral  77% Neutral  93% Neutral  56% Neutral  rs61732181  13399928  C > T  HS3ST3A1  Exon 2 - Synonymous  88% Neutral  58% Deleterious  90% Neutral  93% Deleterious  93% Neutral  56% Neutral  rs61744056  13400057  T > C  HS3ST3A1  Exon 2 - Synonymous  91% Neutral  94% Neutral  97% Neutral  90% Neutral  93% Neutral  62% Deleterious  rs8080565  13400153  G > A  HS3ST3A1  Intron  77% Neutral  86% Neutral  54% Deleterious  85% Neutral  80% Neutral  58% Neutral  rs3744337  13504665  A > G  HS3ST3A1  5′UTR  74% Neutral  67% Deleterious  62% Neutral  93% Neutral  83% Neutral  64% Deleterious  rs3744335  13504884  T > G  HS3ST3A1  5′UTR  88% Neutral  86% Neutral  80% Neutral  93% Neutral    53% Neutral  rs28470223  13505023  A > G  HS3ST3A1  5′UTR  88% Neutral  76% Neutral  86% Neutral  91% Neutral  67% Deleterious  64% Deleterious  rs78863672  13505237  C > A  HS3ST3A1  5′UTR  91% Deleterious  86% Deleterious  62% Neutral  86% Deleterious  67% Deleterious  84% Deleterious  rs2072243  14204380  C > T  HS3ST3B1  5′UTR  97% Deleterious  76% Deleterious  89% Deleterious    68% Deleterious  80% Deleterious  rs2072242  14204410  T > C  HS3ST3B1  5′UTR  97% Deleterious  98% Deleterious  89% Deleterious  88% Deleterious  62% Deleterious  78% Deleterious  rs115229628  14204423  G > A  HS3ST3B1  5′UTR  97% Deleterious  98% Deleterious  100% Deleterious  91% Deleterious  67% Deleterious  76% Deleterious  rs62636623  14205082  G > C  HS3ST3B1  Exon 1 - Missense Gly > Arg  89% Neutral  94% Neutral  90% Neutral  77% Neutral  84% Neutral    rs62636622  14205168  G > A  HS3ST3B1  Exon 1 - Synonymous  96% Neutral  92% Neutral  90% Neutral  87% Neutral  93% Neutral  54% Neutral  rs62056073  14248376  A > G  HS3ST3B1  Exon 2 - Missense Ile > Val  87% Deleterious  58% Neutral  60% Deleterious  59% Deleterious  62% Neutral  51% Deleterious  rs9906855  14248423  T > C  HS3ST3B1  Exon 2 - Synonymous  96% Neutral  94% Neutral  97% Neutral  90% Neutral  93% Neutral  62% Deleterious  rs61729712  14248596  G > A  HS3ST3B1  Exon 2 - Missense Ser>Asn  87% Deleterious  53% Deleterious  60% Deleterious  82% Deleterious  62% Neutral  51% Deleterious  rs55688668  14248702  G > A  HS3ST3B1  Exon 2 - Synonymous  93% Neutral  83% Neutral  90% Neutral  57% Deleterious  93% Neutral  56% Neutral  rs9906590  14248877  G > A  HS3ST3B1  Exon 2 - Missense Glu > Lys  87% Deleterious  53% Deleterious  60% Deleterious  65% Deleterious  62% Neutral  50% Deleterious  rs3785655  14249167  C > T  HS3ST3B1  3′UTR  88% Neutral  86% Neutral  68% Neutral  75% Neutral  83% Neutral  53% Neutral  rs73979332  14249433  C > T  HS3ST3B1  3′UTR  88% Neutral  80% Neutral  62% Neutral  89% Neutral  81% Neutral  71% Neutral  SNP rs28470223 regulates transcriptional promoter activity of HS3ST3A1 We further used bioinformatics to investigate the regulatory effect of the SNP rs28470223 (C > T) associated with a malaria phenotype (Atkinson et al. 2012). The location of rs28470223 within the promoter suggested that this SNP is a cis-regulatory variant controlling HS3ST3A1 gene expression. We first mapped the peaks from ChIP-seq experiments covering a total of 485 transcription factors using the Remap atlas (Griffon et al. 2015; Chèneby et al. 2018) and found 56 transcription factors that bind to rs28470223-containing genomic sequences (Supplementary data, Table S1), indicating a specific regulatory region. We further assessed a possible regulatory functional effect of the SNP rs28470223 on the binding sites of transcription factors using the perfectos-ape tool (Vorontsov et al. 2015) based on the hocomoco v11 motif database (Kulakovskiy et al. 2018). The SNP rs28470223 alters binding of several transcription factors (Supplementary data, Table S2), predicting a functional effect of this SNP in modifying transcription factor binding affinities to rs28470223-containing genomic sequences. The P-value decreases more than 10-fold for the allele T compared with the allele C for the AP2D and KAISO transcription factors whereas it increases more than 10-fold for the BRAC and TCFP2 transcription factors (Supplementary data, Table S2). Finally, the SNP rs28470223 was found to be an expression quantitative trait locus (eQTL) (P = 3.06 10−6) in the Genotype-Tissue Expression database (Consortium 2013). Overall, the bioinformatics analysis supports a cis-regulatory effect of the SNP rs28470223. We next performed gene reporter assays to evaluate the functional effect of the SNP rs28470223 on the HS3ST3A1 promoter activity in K562 cells. As a result, the minor allele (rs28470223-T) significantly decreases the promoter activity by ~54% compared with the wild type allele (rs28470223-C) on the basis of six experiments including both alleles (P = 6 10−5) (Figure 1), confirming the regulatory function of rs28470223 on the promoter transcriptional activity of HS3ST3A1. Fig. 1. View largeDownload slide rs28470223 polymorphism alters HS3ST3A1 promoter activity. Six luciferase reporter gene assays with constructs containing the rs28470223-C (n = 6) or rs28470223-T (n = 6) HS3ST3A1 promoter in K562 cell line were performed. All constructs were co-transfected with pRL-SV40 to standardize transfection efficiency and luciferase activity for each sample was adjusted by the empty pGL3-Enhancer vector. Statistical analysis was performed using two-tailed Student’s t-test after controlling the normality of the data and the equality of their variance. Mean and standard error mean of relative luciferase activity are shown for each allele. Fig. 1. View largeDownload slide rs28470223 polymorphism alters HS3ST3A1 promoter activity. Six luciferase reporter gene assays with constructs containing the rs28470223-C (n = 6) or rs28470223-T (n = 6) HS3ST3A1 promoter in K562 cell line were performed. All constructs were co-transfected with pRL-SV40 to standardize transfection efficiency and luciferase activity for each sample was adjusted by the empty pGL3-Enhancer vector. Statistical analysis was performed using two-tailed Student’s t-test after controlling the normality of the data and the equality of their variance. Mean and standard error mean of relative luciferase activity are shown for each allele. A missense SNP is located within the catalytic domain of 3-OST-3 at proximity of the reducing end of the substrate The catalytic domain of human 3-OST-3B1 shares 99% sequence identity with that of 3-OST-3A1 for which the structure has been determined, arguing that this functionally important region is actively maintained by gene conversion between the 3-OST-3A and 3-OST-3B loci; each gene being duplicated in human (Shworak et al. 1999). The catalytic domain of 3-OST-3 displays an overall α/β motif typically found in sulfotransferases with a large open cleft running across the active site. Central to this structural motif are the phosphosulfate binding loop (P-loop like motif, Lys162-Arg166) of the high-energy sulfate donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) and a conserved glutamate acting as the catalytic base, a binding site well conserved in sulfotransferases (Liu et al. 2012). The acceptor site for sulfonation, referred as the acceptor sugar, lies at the center of the cleft and corresponds to the position of a GlcNS6S residue (T-2 in 3-OST-3/H-3 in 3-OST-1) (Figure 2) (Moon et al. 2004, 2012). Fig. 2. View largeDownload slide Mapping residues corresponding to missense SNP polymorphism on the crystal structure of 3-OST-3A1. (A) Overall view of the catalytic domain of the homologous 3-OST-3 dimer with each subunit colored in cyan and yellow. Mutated residues (orange) that severely affect the catalytic activity are mapped onto the solvent-accessible surface of human 3-OST-3 (accession code 1T8U) in complex with PAP (orange with magenta phosphate) and a tetrasaccharide (blue with green sulfate); those that correspond to the three 3-OST-3B1 SNPs are shown in red with the Ile196Val rs62056073 variant located on the back face. The Ile288Ala mutant, which is closely located to the Ser269Asn variant and less severely affects the catalytic activity, is shown in violet. (B) Close-up of boxed area, oriented as in panel A, of the active site of 3-OST-3 with bound PAP and tetrasaccharide. Fig. 2. View largeDownload slide Mapping residues corresponding to missense SNP polymorphism on the crystal structure of 3-OST-3A1. (A) Overall view of the catalytic domain of the homologous 3-OST-3 dimer with each subunit colored in cyan and yellow. Mutated residues (orange) that severely affect the catalytic activity are mapped onto the solvent-accessible surface of human 3-OST-3 (accession code 1T8U) in complex with PAP (orange with magenta phosphate) and a tetrasaccharide (blue with green sulfate); those that correspond to the three 3-OST-3B1 SNPs are shown in red with the Ile196Val rs62056073 variant located on the back face. The Ile288Ala mutant, which is closely located to the Ser269Asn variant and less severely affects the catalytic activity, is shown in violet. (B) Close-up of boxed area, oriented as in panel A, of the active site of 3-OST-3 with bound PAP and tetrasaccharide. Given that substrate specificity in HS O-sulfotransferases could be determined by residues distal to the catalytic site, we hypothesized that the three naturally occurring 3-OST-3 SNP isoforms would display differential 3-O-sulfotransferase activities compared with the wild type enzyme. To investigate potential functional effects of the coding SNPs on the 3-OST-3 catalytic activity, we mapped the three missense SNPs onto the crystal structure of 3-OST-3 in ternary complex with PAP and a tetrasaccharide (Moon et al. 2004). This structural comparison was extended to the crystal structure of 3-OST isoform 1 in ternary complex with PAP and a heptasaccharide (Moon et al. 2012). The three missense SNPs are located in loop regions at the surface and hardly alter enzyme functionality (Figure 2). Yet, the Ser269Asn SNP is located at the tip of helix α8 and faces the entry of the large substrate binding at site T-4/H-5 on the reducing end at proximity of the GlcNS6S N-sulfate group (tetrasaccharide complex in 3-OST-3) or 6-O-sulfate group (heptasaccharide complex in 3-OST-1). Sequence analysis of a subset of the 3-O-sulfotransferase family members reveals that Ser269 is conserved across mammalian 3-OST-3 orthologs but variability is tolerated at this position in other 3-OST-3 isoforms (Asn in 3-OST-2 and Lys in 3-OST-1/5). The Ser269Asn SNP variant exhibits a slightly higher catalytic activity We expressed and purified human wt 3-OST-3B1 and the Ser269Asn variant using a mammalian cell expression system and anti-FLAG affinity purification tag. Expression of wt 3-OST-3B1 and the Ser269Asn variant clearly shows a band at 37 kDa by western blot analysis corresponding to the recombinant proteins. We used an HS-derived octasaccharide substrate (HS8) as a mimic of a natural HS substrate to identify with a high pressure liquid chromatography (HPLC)-based assay any differences in enzyme activities between the wild type and the Ser269Asn variant. HS8 was prepared by depolymerization of HS with heparinase III, an enzyme that specifically cleaves GlcNAc-containing disaccharide units, thereby releasing intact NS-domain-like oligosaccharides. Digestion products were purified using combined size-exclusion and anion-exchange chromatography to yield size and charge homogenous oligosaccharide species (Pye et al. 1998). Amongst these, HS8 was selected for the 3-OST enzymatic assay as this oligosaccharide displays an average sulfation content suitable for 3-OST-3B1 activity (Supplementary data, Figure S1). HS8 was incubated with wt 3-OST-3B1 or the Ser269Asn variant and reaction was monitored by measuring the disaccharide composition using a dedicated HPLC-based assay (see Materials and methods). HPLC analysis of disaccharides present in untreated HS8 yielded four major peaks, enabling determination of the disaccharide composition after integration and normalization of the peak areas (Supplementary data, Table S3). In contrast, a similar disaccharide analysis of 3-OST-3B1-treated HS8 showed the appearance and time-dependent increase of an unknown, late-eluting saccharide species (Figure 3). This species most likely corresponds to a heparinase-resistant 3-O-sulfated tetrasaccharide, as previously reported (Yamada et al. 1993). Concomitantly, the 3-OST-3B1 activity decreased the ΔHexA,2S-GlcNS disaccharide content while the pattern of other HS8 disaccharides (i.e. ΔHexA,GlcNAc, ΔHexA,GlcNS and ΔHexA,2S-GlcNS6S) remained unchanged (Figure 3 and Supplementary data, Figure S2). These data clearly indicate that the 3-OST-3B1-caltalyzed 3-O-sulfation event on HS8 is non-random and may preferentially target IdoA2S-GlcNS units, in agreement with previous studies and on the fact that the 3-OST-3 modification must precede the 6-O-sulfation step (Liu, Shworak, et al. 1999; Thacker et al. 2014; Wang et al. 2017). Instead, the 3-OST-1 modification occurs only after 6-O-sulfation to generate the -GlcA,GlcNS3S6S- disaccharide unit (Wang et al. 2017). Fig. 3. View largeDownload slide Analysis of 3-OST-3B1-catalyed 3-O-sulfation of HS8 oligosaccharide. (A) RPIP-HPLC-elution profiles of untreated (gray trace) and 3-OST-3B1-treated (black trace) HS8 oligosaccharides. The peak (marked *) eluted at the highest NaCl concentration corresponds to a new 3-OST-3B1-catalyzed 3-O-sulfated species. (B) Time course of 3-O-sulfation of HS8 by wt 3-OST-3B1 (open circles) and the Ser269Asn mutant (closed circles) showing the evolution of the disaccharide content in ΔHexA,GlcNAc (left), unknown species (peak *, middle) and ΔHexA,2S-GlcNS (right). ΔHexUA-GlcNAc (left) and ΔHexUA,2S-GlcNS (right) contents have been normalized and expressed as a percentage of all disaccharides detected. The amount of the * species (middle) has been normalized using arbitrary units and expressed as a percentage of the total fluorescence signal detected. Fig. 3. View largeDownload slide Analysis of 3-OST-3B1-catalyed 3-O-sulfation of HS8 oligosaccharide. (A) RPIP-HPLC-elution profiles of untreated (gray trace) and 3-OST-3B1-treated (black trace) HS8 oligosaccharides. The peak (marked *) eluted at the highest NaCl concentration corresponds to a new 3-OST-3B1-catalyzed 3-O-sulfated species. (B) Time course of 3-O-sulfation of HS8 by wt 3-OST-3B1 (open circles) and the Ser269Asn mutant (closed circles) showing the evolution of the disaccharide content in ΔHexA,GlcNAc (left), unknown species (peak *, middle) and ΔHexA,2S-GlcNS (right). ΔHexUA-GlcNAc (left) and ΔHexUA,2S-GlcNS (right) contents have been normalized and expressed as a percentage of all disaccharides detected. The amount of the * species (middle) has been normalized using arbitrary units and expressed as a percentage of the total fluorescence signal detected. A kinetic analysis of the Ser269Asn variant activity compared with wt 3-OST-3B1 revealed similar elution profiles, with a slightly faster increase of the peak * area associated to a concomitant faster decrease of the ΔHexA,2S-GlcNS disaccharide content, as monitored by a time course treatment (Figure 3). This result points to a slightly higher catalytic activity for the Ser269Asn variant compared with the wt enzyme. In fact, structural analysis suggests that a bulkier Asn in the 3-OST-3B1 variant could provide additional polar interactions with the N- or 6-O-sulfate group of GlcNS6S at site T-4 near the reducing end of the substrate compared with a shorter Ser residue in wt 3-OST3. Site-directed mutagenesis of the adjacent Trp268 residue in 3-OST-3, which contributes to a sulfate-binding pocket near the reducing end of the substrate, resulted in a 41-fold reduction in catalytic efficiency, arguing for a functional role of the contiguous Ser269 in substrate binding (Moon et al. 2012). In addition to Ser269, other residues remote from the active site but located at the reducing end of the substrate, including Thr256 in 3-OST-3 and Arg268 in 3-OST-1, were shown to play important roles in substrate binding and specificity between 3-OST isoforms (Moon et al. 2012). Overall, this first genetic and biochemical analyses of two genetic variants in the HS3ST3A1/B1 genes associated with P. falciparum parasitaemia offer first insights into the modulation of 3-OST-3 gene transcription and catalytic activity by SNPs. Whether the SNPs affected HSPG level and structure in the population living in an endemic area in Burkina Faso is unknown and merits further investigation. Materials and methods In silico analysis of the SNP regulatory effect We explored ChIP-seq results by using the ReMap atlas covering 485 transcription factors to identify transcription factors that bind DNA sequence containing the studied SNPs (Chèneby et al. 2018). To this aim, we used the ReMap tool to intersect the genomic coordinates of SNPs with those of the ChIP-seq peaks. Furthermore, we used the PERFECTOS-APE software to predict the effect of the SNPs on the binding of transcription factors (Vorontsov et al. 2015). Briefly, the extracted SNPs sequences and their alleles together with their flanking regions were scanned against a recent collection of motifs, which contains binding models of 680 human transcription factors (Kulakovskiy et al. 2018). The site score was obtained from position-specific scoring matrices on sequences containing each allele, allowing us to assess the P-value for each allele and therefore the P-value ratio. The candidate transcription factors were retained on the basis of the best P-value obtained for one of the allele; it should be lower than 10−3. The calculated P-value ratio reflects the predicted effect of the SNP on the binding of the transcription factor on the SNP-containing DNA sequence. Transient transfection and dual-luciferase reporter assay K562 cells (ATCC CLL-243) were grown in Gibco RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS). A 1200 bp DNA fragment upstream the HS3ST3A1 translation start site (chromosome 17:13504472-13505672 according to the hg38 assembly) was cloned by gene synthesis (GeneCust Custom Services, Luxembourg). This fragment was cloned into the MluI-XhoI sites of the pGL3-basic vector (Promega, Madison, WI, USA), which contains the firefly luciferase coding sequence. Initially, the pGL3 construct contained the rs28470223 T-allele which represents the minor allele in Africa, whereas the rs28470223-C is the major allele in Africa and is considered as the wild type allele. To generate the pGL3 construct containing the rs28470223-C allele, site-directed mutagenesis was performed with the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA) using primers (5′-AGGTTCTCTCGGCCAAGGAGC-3′, 3′-GAACTCGGGCGGAGAGAA-5′) designed by NEBaseChanger tool and provided by the supplier, and an annealing temperature of 63°C. K562 transfection was performed with the Neon™ Transfection System (Invitrogen) following manufacturer’s instructions. For each of the six tests performed, 106 cells were co-transfected with 1 μg of the empty pGL3-basic control vector, or with positive pGL3-promoter control vector, or with 1 μg of pGL3-basic vector containing the rs28470223-T (p-T) or rs28470223-C (p-C) HS3ST3A1 promoter sequence together with 200 ng of the pRL-SV40 plasmid encoding Renilla luciferase (Promega, Madison, WI, USA), which was used as a transfection efficiency control. Transfected cells were maintained at 37°C in 5% CO2 during 24 h. Firefly and Renilla luciferase activities were monitored using a TriStar LB 941 Multimode Microplate Reader (Berthold technologies, Thermo Fisher Scientific, Waltham, MA, USA) by analyzing 20 μl of cell lysate according to the manufacturer’s instructions provided in the Dual-Luciferase Kit (Promega). The firefly luciferase activity of each sample was normalized by the Renilla luciferase activity and adjusted by the pGL3-basic mean activity. Statistical analysis To assess the effect of SNPs on the promoter activity, Student’s t-test was used after checking the normality of the distributions by using the Shapiro–Wilk method and their variance equality by using a Fisher test. All analyses were performed by using either R or SPSS software (SPSS, Boulogne, France). Only significant terms at the 5% level were retained. Expression and purification of human 3-OST-3B1 and the Ser269Asn variant Following unsuccessful attempts to express a soluble form of the human 3-OST-3B1 catalytic domain (Ile125-Asp390) in Escherichia coli, either as a 6xHis- or thioredoxine-tagged form, a cDNA fragment encoding the catalytic domain of wt 3-OST-3B1 was cloned into the pYD7 vector suitable for expression in human embryonic kidney HEK293 EBNA cell line. The recombinant pYD7-3-OST-3B1 vector consists of a Kozak consensus sequence, a SEAP peptide signal and a Flag tag upstream of the catalytic domain for 3-OST-3B1 detection and purification. The pYD7 plasmid carrying the 3-OST-3B1 Ser269Asn variant (rs61729712) was amplified by polymerase chain reaction (PCR) from the wild type construct using the forward 5′-TCGACACGTCGTGGAACGCCATCCAGATCGG-3′ and reverse 5′-CCGATCTGGATGGCGTTCCACGACGTGTCGA-3′ oligonucleotides. The PCR product was cloned into the linearized PYD7 plasmid DNA and the sequence was verified by sequencing. Plasmid DNA (2 μg) was transfected with 4 μg of linear polyethylenimine (Polysciences) into HEK293 EBNA cells in a 6-well culture plate. Transfected cells cultured in Dulbecco's modified Eagle's medium (DMEM) 10% FBS were selected with blasticidin (5 μg/ml) for stable cell line establishment. Higher expression and secretion levels of the protein produced by the cell lines were achieved in DMEM 2% FBS, 1.25 mM sodium valproate (Sigma-Aldrich) and 0.5% Tryptone N1 (Organotechnie). Protein production was performed in multilayer culture flasks at 32°C instead 37°C to reduce protein aggregation. Protein secretion was analyzed by western blot using an anti-FLAG tag antibody. Before purification, culture medium containing secreted protein was harvested after 7 days of culture and dialyzed overnight at 4°C against 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 8.0 and 100 mM NaCl. FLAG-tagged wt 3-OST-3B1 was purified by anti-FLAG M2 affinity (Sigma-Aldrich) and eluted in a buffer containing 20 mM HEPES pH 8.0, 100 mM NaCl and 100 μg/ml FLAG tag peptide (Sigma-Aldrich). Fractions containing 3-OST-3B1 were pooled and further purified by size-exclusion chromatography on a Superdex 200 26/60 column (GE-Healthcare) equilibrated with 20 mM Tris pH 8.0 and 200 mM NaCl. The Ser269Asn variant was produced and purified as for wt 3-OST-3B1. Protein purity and integrity of wt 3-OST-3B1 and the Ser269Asn variant were analyzed by SDS-PAGE electrophoresis and matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry, and concentrated by ultrafiltration to 1 mg/ml in 20 mM Tris-HCl pH 8.0, 100 mM NaCl and 1 mM PAP (Sigma-Aldrich). Preparation of HS-derived octasaccharides Porcine mucosal HS (Celsius) was extensively digested with 25 mU/ml of heparinase III (Grampian Enzyme) in 5 mM Tris-HCl, 2 mM CaCl2, 0.1 mg/ml BSA, 50 mM NaCl, pH 7.5 for 72 h at 30°C. The resulting mixture was resolved onto a 1500 mm × 44 mm Biogel P10 column (Bio-Rad) in 200 mM NaCl at 1 ml/min, leading to a graded series of size-uniform oligosaccharides from disaccharide to octadecasaccharide. Samples were dialyzed against distilled water, freeze-dried and quantified. The octasaccharides were further purified by strong-anion-exchange HPLC, on a 9 × 250 mm preparative ProPac PA1 column (Dionex). After equilibration in mobile phase (distilled water adjusted to pH 3.5 with HCl) at 1 ml/min, samples (7.5 mg) were injected and eluted with a gradient of NaCl (0–0.5 M over 10 min, then 0.5–1 M over 80 min) in the same mobile phase. The eluate was monitored online for UV absorbance at 232 nm, and 23 different fractions were collected, dialyzed against distilled water, freeze-dried, quantified and analyzed as described below. Fraction HS8 was used as a substrate to monitor the 3-OST-3B1 catalytic activity. Time course of 3-O-sulfation of the HS8 oligosaccharide by 3-OST-3B1 Lyophilized HS8 was resuspended in 50 mM MES pH 7.0, 5 mM MgCl2, 200 μM PAPS and incubated with 1 μg of 3-OST-3B1 (wild type or the Ser269Asn variant) for 1 h at 37°C. For time course digestion, aliquots were taken off at 0, 1, 3, 6 and 24 h time points in triplicate. Samples were then boiled for 5 min to inactive the enzyme and then stored at −20°C. Disaccharide analysis Disaccharide analysis of the HS8 samples was performed as previously described (Henriet et al. 2017). Briefly, samples in 100 mM sodium acetate, 0.5 mM calcium acetate, pH 7.1 were digested into disaccharides by incubation with a cocktail of heparinase I, II and III (10 mU each) overnight at 37°C. These experimental conditions are not suitable for the 3-O-sulfation specific peeling reaction. Disaccharide composition was determined by RPIP-HPLC, by injection on a Luna 5 μm C18 reversed phase column (4.6 × 300 mm, Phenomenex, Le Pecq, France) equilibrated at 0.5 ml/min in 1.2 mM tetra-N-butylammonium hydrogen sulfate (TBA) in 8.5% acetonitrile. Disaccharides were then resolved using a multi-step NaCl gradient (0–30 mM in 1 min, 30–90 mM in 39 min, 90–228 mM in 2 min, 228 mM for 4 min, 228–300 mM in 2 min, 300 mM for 4 min). Online post-column disaccharide derivatization was achieved by addition of 2-cyanoacetamide (0.25%) in NaOH (0.5%) at a flow rate of 0.16 ml/min, followed by fluorescence detection (excitation 346 nm, emission 410 nm). Fluorescence signal was normalized with HS disaccharide standards (Iduron, Alderley Edge, UK) or using arbitrary units for the unknown (peak *) saccharide species in absence of standards. Direct comparison of these normalized disaccharide contents is possible, unlike that of the unknown (peak *) species. Structural analysis A structural analysis was performed to map the three 3-OST-3B1 missense SNPs on the crystal structure of 3-OST-3 bound to PAP and a tetrasaccharide substrate (accession code 1T8U) or 3-OST-1 bound to PAP and heptasaccharide substrate (3UAN). The root-mean-square deviation value between the two structures is 0.95 Å for 244Cα atoms (44% sequence identity). Supplementary data Supplementary data is available at GLYCOBIOLOGY online. Funding This work was supported in part by the CNRS, INSERM and Aix-Marseille University to P.R./Y.B., the GDR GAG (GDR 3739) to R.R.V./H.L-J., the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-05-01 to Y.B./H.L-J., the LabEx GRAL (ANR-10-LABX-49-01) and the “Investissements d’avenir” program Glyco@Alps (ANR-15-IDEX-02) to H.L-J./R.R.V. Acknowledgements TNN was supported by a PhD fellowship from the Vietnamese government. We thank Melanie Daligault (TAGC, Marseille) for helpful assistance with bioinformatics analyses, Ahmad Ali-Ahmad and Pascale Marchot (AFMB, Marseille) for helpful assistance with biochemical analyses. Conflict of interest statement None declared. 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GlycobiologyOxford University Press

Published: Apr 28, 2018

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