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Posttranslational Protein Modification: Biosynthetic Control Mechanisms in the Glycosylation of the Major Myelin Glycoprotein by Schwann Cells
Author(s) -
Poduslo Joseph F.
Publication year - 1985
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1985.tb08743.x
Subject(s) - concanavalin a , mannose , oligosaccharide , biochemistry , glycoprotein , myelin , chemistry , lectin , affinity chromatography , fucose , glycosylation , myelin associated glycoprotein , glycopeptide , gel electrophoresis , microbiology and biotechnology , in vitro , biology , enzyme , central nervous system , neuroscience , antibiotics
The posttranslational processing of the asparagine‐linked oligosaccharide chain of the major myelin glycoprotein (P 0 ) by Schwann cells was evaluated in the permanently transected, adult rat sciatic nerve, where there is no myelin assembly, and in the crush injured nerve, where there is myelin assembly. Pronase digestion of acrylamide gel slices containing the in vitro labeled [ 3 H]mannose and [ 3 H]fucose P 0 after electrophoresis permitted analysis of the glycopeptides by lectin affinity and gel filtration chromatography. The concanavalin A‐Sepharose profile of the [ 3 H]mannose P 0 glycopeptides from the transected nerve revealed the high‐mannose‐type oligosaccharide as the predominant species (72.9%), whereas the normally expressed P 0 glycoprotein that is assembled into the myelin membrane in the crushed nerve contains 82.9–91.9% of the [ 3 H]mannose radioactivity as the complex‐type oligosaccharide chain. Electrophoretic analysis of immune precipitates verified the [ 3 H]mannose as being incorporated into P 0 for both the transected and crushed nerve. The high‐mannose‐type glycopeptides of the transected nerve isolated from the concanavalin A‐Sepharose column were hydrolyzed by endo‐β‐ N ‐acetylglucosaminidase H, and the oligosaccharides were separated on Biogel P4. Man 8 GlcNAc and Man 7 GlcNAc were the predominant species with radioactivity ratios of 12.5/7.2/1.4/1.0 for the Man 8 , Man 7 , Man 6 , and Man 5 oligosaccharides, respectively. Jack bean α‐ d ‐mannosidase gave the expected yields of free Man and ManGlcNAc from these high‐mannose‐type oligosaccharides. The data support the notion that at least two α‐1,2‐mannosidases are responsible for converting Man 9 GlcNAc 2 to Man 5 GlcNAc 2 . The present experiments suggest distinct roles for each mannosidase and that the second mannosidase (I‐B) may be an important rate‐limiting step in the processing of this glycoprotein with the resulting accumulation of Man 8 GlcNAc 2 and Man 7 GlcNAc 2 intermediates. Pulse chase experiments, however, demonstrated further processing of this highmannose‐type oligosaccharide in the transected nerve. The [ 3 H]mannose P 0 glycoprotein with M r of 27,700 having the predominant high‐mannose‐type oligosaccharide shifted its M r to 28,500 with subsequent chase. This band at 28,500 was shown to have the complex‐type oligosaccharide chain and to contain fucose attached to the core asparagine‐linked GlcNAc residue. The extent of oligosaccharide processing of this down‐regulated glycoprotein remains to be determined.