Functional Characterization of Glycosylation-Deficient Human P-Glycoprotein Using A Vaccinia Virus Expression System

Functional Characterization of Glycosylation-Deficient Human P-Glycoprotein Using A Vaccinia... P-glycoprotein (P-gp), the product of human MDR1 gene, which functions as an ATP-dependent drug efflux pump, is N-linked glycosylated at asparagine residues 91, 94, and 99 located within the first extracellular loop. We report here the biochemical characterization of glycosylation-deficient (Gly−) P-gp using a vaccinia virus based transient expression system. The staining of HeLa cells expressing Gly− P-gp (91, 94, and 99N→Q), with P-gp specific monoclonal antibodies, MRK-16, UIC2 and 4E3 revealed a 40 to 50% lower cell-surface expression of mutant P-gp compared to the wild-type protein. The transport function of Gly− P-gp, assessed using a variety of fluorescent compounds indicated that the substrate specificity of the pump was not affected by the lack of glycosylation. Additional mutants, Gly− D (91, 94, 99N→D) and Gly−Δ (91, 94, 99 N deleted) were generated to verify that the reduced cell surface expression, as well as total expression, were not a result of the glutamine substitutions. Gly− D and Gly−Δ Pgps were also expressed to the same level as the Gly− mutant protein. 35S-Methionine/cysteine pulse-chase studies revealed a reduced incorporation of 35S-methionine/cysteine in full length Gly− P-gp compared to wild-type protein, but the half-life (∼3 hr) of mutant P-gp was essentially unaltered. Since treatment with proteasome inhibitors (MG-132, lactacystin) increased only the intracellular level of nascent, mutant P-gp, the decreased incorporation of 35S-methionine/cysteine in Gly− P-gp appears to be due to degradation of improperly folded mutant protein by the proteasome and endoplasmic reticulum-associated proteases. These results demonstrate that the unglycosylated protein, although expressed at lower levels at the cell surface, is functional and suitable for structural studies. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Functional Characterization of Glycosylation-Deficient Human P-Glycoprotein Using A Vaccinia Virus Expression System

Loading next page...
 
/lp/springer_journal/functional-characterization-of-glycosylation-deficient-human-p-qOrmjcm6B8
Publisher
Springer Journals
Copyright
Copyright © Inc. by 2000 Springer-Verlag New York
Subject
Life Sciences; Biochemistry, general; Human Physiology
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s002320001020
Publisher site
See Article on Publisher Site

Abstract

P-glycoprotein (P-gp), the product of human MDR1 gene, which functions as an ATP-dependent drug efflux pump, is N-linked glycosylated at asparagine residues 91, 94, and 99 located within the first extracellular loop. We report here the biochemical characterization of glycosylation-deficient (Gly−) P-gp using a vaccinia virus based transient expression system. The staining of HeLa cells expressing Gly− P-gp (91, 94, and 99N→Q), with P-gp specific monoclonal antibodies, MRK-16, UIC2 and 4E3 revealed a 40 to 50% lower cell-surface expression of mutant P-gp compared to the wild-type protein. The transport function of Gly− P-gp, assessed using a variety of fluorescent compounds indicated that the substrate specificity of the pump was not affected by the lack of glycosylation. Additional mutants, Gly− D (91, 94, 99N→D) and Gly−Δ (91, 94, 99 N deleted) were generated to verify that the reduced cell surface expression, as well as total expression, were not a result of the glutamine substitutions. Gly− D and Gly−Δ Pgps were also expressed to the same level as the Gly− mutant protein. 35S-Methionine/cysteine pulse-chase studies revealed a reduced incorporation of 35S-methionine/cysteine in full length Gly− P-gp compared to wild-type protein, but the half-life (∼3 hr) of mutant P-gp was essentially unaltered. Since treatment with proteasome inhibitors (MG-132, lactacystin) increased only the intracellular level of nascent, mutant P-gp, the decreased incorporation of 35S-methionine/cysteine in Gly− P-gp appears to be due to degradation of improperly folded mutant protein by the proteasome and endoplasmic reticulum-associated proteases. These results demonstrate that the unglycosylated protein, although expressed at lower levels at the cell surface, is functional and suitable for structural studies.

Journal

The Journal of Membrane BiologySpringer Journals

Published: Feb 1, 2000

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off