ORIGINAL ARTICLE
Degradation of glycosphingolipids in oyster: ceramide glycanase
and ceramidase in the hepatopancreas of oyster,
Crassostrea virginica
Nadejda V. Pavlova
1
&
Su-Chen Li
1
&
Yu-Teh Li
1
Received: 14 August 2017 /Revised: 3 October 2017 /Accepted: 4 October 2017 /Published online: 16 October 2017
#
Springer Science+Business Media, LLC 2017
Abstract The hepatopancreas of oyster, Crassostrea
virginica, was found to contain two unique glycosphingolipid
(GSL) cleaving enzymes, ceramide glycanase (CGase) and
ceramidase. These two enzymes were found to be tightly as-
sociated together through the consecutive purification steps
including gel filtration, hydrophobic interaction and cation-
exchange chromatographies. They were separated only by
preparatory SDS-PAGE. The purified CGase was found to
have a molecular mass of 52 kDa and pH optimum of 3.2–
3.3. This enzyme prefers to hydrolyze the acidic GSLs,
II
3
SO
3
LacCer and gangliosides over the neutral GSLs.
Oyster ceramidase was found to have a molecular mass of
88 kDa and pH optimum of 4–4.5. Since oyster ceramidase
greatly prefers ceramides with C
6
to C
8
fatty acids, C
6
-cer-
amide (N-hexanoyl-D-sphingosine) was used as the substrate
for its purification and characterization. The oyster acid
ceramidase also catalyzed the synthesis of ceramide from a
sphingosine and a fatty acid. For the synthesis, C
16
and C
18
fatty acids were the best precursors. The amino acid sequences
of the two cyanogenbromide peptides derived from the puri-
fied ceramidase were found to have similarities to those of
several neutral and alkaline ceramidases reported. The tight
association of CGase and ceramidase may indicate that CGase
in oyster hepatopancreas acts as a vehicle to release ceramide
from GSLs for subsequent generation of sphingosines and
fatty acids by ceramidase to serve as signaling factors and
energy source.
Keywords Oyster
.
CGase
.
Ceramidase
.
Glycolipids-cleaving enzyme
Introduction
As oysters are filter feeders, their digestive enzymes must be
able to degrade the ingested organisms having the varieties of
biomolecules including glycosphingolipids (GSLs). The ca-
tabolism of a GSL involves the conversion of the hydrophilic
sugar chain into monosaccharides and the hydrophobic cer-
amide moiety into sphingosine and fatty acid. In higher ani-
mals, exo-glycosidases are responsible for the stepwise deg-
radation of the sugar chain in a GSL to expose the ceramide
that in turn is converted into sphingosine and fatty acid [1]. In
addition to this exo-glycosidase pathway, a new catabolic
pathway for GSLs has been revealed by the discovery of cer-
amide glycanase (CGase) in annelids [2, 3]. The first step of
this new pathway is the cleavage of the linkage between the
sugar chain and the ceramide in GSLs by CGase. The sugar
chain and the ceramide are further separately catabolyzed by
exo-glycosidases and ceramidase, respectively. In the animal
kingdom, CGase has been found in leech [2], earthworm [3],
clam [4], jellyfish [5] and hydrozoa [6]. CGase was also
named endoglycoceramidase (EGCase) and has been induced
in Rhodococcus [7]. The presence or absence of this unique
GSL-cleaving activity in mammalian tissues is still not settled
[8]. One of the tantalizing yet important questions is the bio-
logical function of CGase. We found that the hepatopancreas
of oyster, Crassostrea virginica, contained two unique GSL-
cleaving enzymes, CGase and ceramidase, but not
sphingomyelinase. The ceramide released from a GSL by
the oyster CGase was subsequently converted into sphingo-
sine and fatty acid by ceramidase. During the purification of
CGase and ceramidase from oyster hepatopancreas, these two
* Yu -Teh L i
yli1@tulane.edu
1
Department of Biochemistry and Molecular Biology, Tulane
University School of Medicine, 1430 Tulane Avenue, New
Orleans, LA 70112, USA
Glycoconj J (2018) 35:77–86
https://doi.org/10.1007/s10719-017-9802-7
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