Abnormal expression and glycosylation of the large and small isoforms of myelin‐associated glycoprotein in dysmyelinating quaking mutants

Abstract

The relative expression of large (L) and small (S) isoforms of the myelin-associated glycoprotein (MAG) and their glycosylation were compared in developing spinal cord of quaking and control mice. Using antisera specific for L- and S-MAG, respectively, it was shown that S-MAG is the principal isoform in quaking mice at all ages between 13 and 72 days, although L-MAG was just detectable by western blotting at the early ages. Both L- and S-MAG have higher apparent molecular weights in quaking mice than in controls. Experiments involving lectin binding and glycosidase treatment demonstrated that the higher molecular weight of MAG in the quaking mutant was due to a higher content of N-acetylneuraminic acid residues linked alpha 2-3 to galactose as well as to more branching of oligosaccharide moieties indicated by a higher content of subterminal galactose residues. The total sialic acid measured by HPAE-chromatography in purified quaking MAG was 40% higher than in control MAG. By contrast, quaking MAG contained less of the adhesion-related, HNK-1 carbohydrate epitope. Another difference was that a lower molecular weight form of MAG with predominantly high mannose oligosaccharides was prominent in young quaking mice, but not in controls. The abnormalities of MAG expression related to splicing of its mRNA and glycosylation may contribute to the myelin pathology in quaking mutants.

Endocytic depletion of L-MAG from CNS myelin in quaking mice.

“…Our ultrastructural studies indicate that periaxonal L-MAG is removed by endocytosis and not replaced. In addition, biochemical studies have demonstrated that both L-and S-MAG are abnormally glycosylated in quaking mutants (Bartoszewicz et al, 1994). Overall, our combined immunocytochemical and biochemical studies indicate that the quaking mutation leads to an under expression of L-MAG that becomes more severe with maturation, abnormalities in the posttranslational glycosylation of MAG, and the eventual depletion of L-MAG from the periaxonal membrane.…”

“…One report indicated that L-MAG was barely detectable in developing and adult quaking mice (Fujita et al, 1990), while another detected equal amounts of L-and S-MAG in adult mice (Braun et al, 1990). To clarify these apparent discrepancies, the present immunocytochemical study was undertaken in parallel with a biochemical investigation of MAG expression in spinal cords from quaking mice (Bartoszewicz et al, 1994). Both approaches detected significant L-MAG in young quaking mice and a dramatic decrease in L-MAG with maturation.…”

“…The level of L-MAG mRNA is dramatically reduced in quaking CNS (Frail et al, 1985;Fujita et al, 1988), while L-MAG has been reported to be either scarcely detectable (Fujita et al, 1990;Bartoszewicz et al, 1994) or present in equal levels to S-MAG (Braun et al, 1990). S-MAG mRNA levels are increased when compared with controls, while the level of S-MAG protein is reduced in quaking, but not to the same degree as L-MAG (Fujita et al, 1990;Fujita et al, 1988;Bartoszewicz et al, 1994). In quaking, the molecular weight of both L-and S-MAG is increased, due to abnormal glycosylation (Matthieu et al, 1974;Bartoszewicz et al, 1994).…”

“…S-MAG mRNA levels are increased when compared with controls, while the level of S-MAG protein is reduced in quaking, but not to the same degree as L-MAG (Fujita et al, 1990;Fujita et al, 1988;Bartoszewicz et al, 1994). In quaking, the molecular weight of both L-and S-MAG is increased, due to abnormal glycosylation (Matthieu et al, 1974;Bartoszewicz et al, 1994).…”

Structure-Function Analysis of Qk1: a Lethal Point Mutation in Mouse quaking Prevents Homodimerization

“…The quaking viable mice have been extensively studied (20), and several defects in RNA metabolism have been observed. Alterations in the levels of alternatively spliced RNAs and in the processing and/or turnover of the mRNA transcripts encoding myelin-associated glycoprotein, myelin basic protein, and proteolipid protein have been demonstrated (4,6,13,19). A defect in myelin basic protein mRNA transport has also been observed in the quaking viable mice (3).…”