Arginyl-Methionine

The PO glycoprotein, the major protein of peripheral nerve myelin, is a hydrophobic glycoprotein which can be isolated in soluble and insoluble forms from rabbit sciatic nerve myelin following extensive defatting and mid acidic extraction. The PO glycoprotein was localized exclusively in peripheral nervous system (PNS) myelin of sciatic nerve and rootlets by the immunofluorescent technique using goat anti-PO serum which showed a single precipitin band in double diffusion and did not cross-react with the myelin basic protein or P2 protein. Central nervous system (CNS) myelin from brain and spinal cord was negative by the immunofluorescent procedure. The major glycoprotein bands in PNS myelin, in addition to the PO glycoprotein at 28K, exist at 23K and 19K, as shown by gel electrophoresis in dodecyl sulfate. These glycoproteins, isolated by gel filtration in 2% dodecyl sulfate, show identity to the PO glycoprotein in their monosaccharide profile and overlapping tryptic peptides on peptide mapping. We conclude that both the 23K and 19K glycoproteins are derived from the PO glycoprotein by in situ proteolysis; the 23K glycoprotein has the identical amino terminal sequence. The 19K glycoprotein, beginning with amino-terminal methionine, is identical with the TPO glycoprotein, shown previously to originate from tryptic hydrolysis of the PO glycoprotein in isolated myelin. A tryptic glycopeptide containing 27 amino acids was isolated from the PO glycoprotein and sequenced. It contained a relatively high proportion of aspartic acid (four residues) and glutamic acid (two residues), thus exhibiting a high negative charge. We conclude that the total carbohydrate of the PO, 23K, and 19K glycoproteins does indeed exist as a single nonasaccharide moiety linked through N-acetylglucosamine to Asp-14 of the glycopeptide in a N-glycosidic linkage. These results further support the role of the PO glycoprotein as a typical amphipathic membrane protein.

When purified rabbit sciatic nerve myelin, whether lyophilized or not, is treated with low amounts of trypsin (25 microgram/ml) for 0.5, 3, or 24 h the resulting protein patterns viewed on sodium dodecyl sulfate (SDS) gel electrophoresis are similar. The most striking feature of the trypsinized myelin is the accumulation of a heavy band at the basic protein position, molecular weight 19 000, which is accounted for as a degradation product of the PO protein, referred to as the TPO protein. The PO protein, the major glycoprotein of sciatic nerve myelin, as well as the 23K and P2 proteins and albumin, an absorbed component, are all partially degraded; most high molecular weight bands are lost. The TPO protein, isolated by gel filtration in 2% SDS on an agarose column, like the PO protein, is highly insoluble in aqueous solvents. It is a glycoprotein (8% carbohydrate), staining with periodic acid-Schiff reagent; containing 3 mannose, 1 galactose, 3 N-acetylglucosamine, 1 sialic acid, and 1 fucose residues and is identical to the nonasaccharide of the parent PO protein. The amino acid composition of the TPO protein, is similar to the PO protein, but has a much higher content of hydrophobic residues and begins with NH2-methionine. This suggests that the PO protein is an amphipathic membrane protein in which its more polar character is confined to the first third of its NH2-terminus. This polar domain is probably positioned above the lipid leaflet where it is accessible to trypsin which cleaves a sensitive lysinyl (or argininyl)-methionine linkage. The more hydrophobic domain (the TPO protein) is buried in the myelin bilayer where it is protected from further tryptic attack. Thus trypsin can serve as a useful probe of myelin structure.

A glycoprotein, referred to as PO protein, was isolated from rabbit sciatic nerve myelin by gel filtration on Agarose 0.5 m in dodecyl sulfate. The purified myelin was first defatted and extracted at pH 2. The water-soluble proteins such as myelin basic protein and P2 protein were extracted leaving a glycoprotein-rich residue, from which the PO protein was isolated. The purified protein showed a single band on gel electrophoresis in dodecyl sulfate when stained with Coomassie Blue of periodic acid-Schiff reagent. The carbohydrate, comprising 6.3% by weight, appears to exist as a nonasaccharide unit having 3 mannose, 3 N-acetylglucosamine, 1 sialic acid, 1 galactose and 1 fucose residue. The polypeptide moiety has a high content of non-polar amino acids. A single amino acid, isoleucine, was found at the NH2-terminal end by dansyl and Edman procedures. The PO protein is the major protein of peripheral nerve myelin.