Keiichi Uyemura

Purification and partial characterization of two glycoproteins in bovine peripheral nerve myelin membrane.

Two major glycoproteins of bovine peripheral nerve myelin were isolated from the acid-insoluble residue of the myelin by a procedure involving delipidation with chloroform/methanol (2:1, v/v) and chromatography on Sephadex G-200 column with a buffer containing sodium dodecyl sulfate. The separation patterns of the proteins on the gel were affected considerably by the dodecyl sulfate concentration in the elution buffer. At above 2% dodecyl sulfate concentration in the elution buffer, the glycoproteins, could be separated clearly on the gel and were purified. The purified proteins, the BR protein (mol. wt. 28 000) and the PAS-II protein (mol. wt. 13 000), were homogeneous on dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2- terminal amino acids of the BR and the PAS-II proteins were isoleucine and methionine, respectively. The BR protein contained glucosamine, mannose, galactose, fucose and sialic acids and the PAS-II protein contained glucosamine, mannose, galactose, fucose and glucose. Neither the BR protein nor the PAS-II were a glycosylated derivative of a basic protein of bovine peripheral nerve myelin, a deduction based on the results of amino acid analysis. The two major glycoproteins were observed commonly in the peripheral nerve myelin of cows, pigs, rabbits and guinea pigs, using dodecyl sulfate-polyacrylamide gel electrophoresis.

The complete amino acid sequence of the P2 protein in bovine peripheral nerve myelin

The P2 protein is a basic protein localizedin peripheral nerve myelin [ 1 ]. We have developed pu~~cation procedures of the P2 protein and characterized some of its properties [2-71. The P2 protein is considered to be the most likely candidate of neuritogenic protein to induce experimental allergic neuritis (EAN). However, our attempts to induce EAN in guinea pig or rabbit by injection of purified bovine P2 protein have been unsuccessful [3,7]. We confirmed the fmding that bovine P2 protein induces EAN in the Lewis rat (891. In order to clarify the antigenic determinant(s) of the P2 protein to induce EAN, determination of primary structure of the protein is essential. Here we report the complete amino acid sequence of bovine P2 protein.

DEVELOPMENTAL CHANGES OF MYELIN PROTEINS IN THE CHICK PERIPHERAL NERVE

Abstract— Developmental changes of myelin proteins in chick sciatic nerve were studied at the stage of myelination by sodium dodecylsulfate (SDS)‐polyacrylamide gel electrophoresis. The myelin of adult hen peripheral nervous system (PNS) contained two glycoproteins (BR‐P0 and PASII), both of which are unique to PNS myelin, in addition to the basic encephalitogenic protein, BP, which is common to CNS and PNS myelin. The other basic protein (BF‐P2) found in the PNS of other species was not definitely detectable in hen PNS. At the early stages of myelination (from 14 to 18 embryonic days) the amounts of myelin proteins increased rapidly in parallel with the increase in number of layers of the myelin sheath of the PNS. At 14 embryonic days high molecular weight proteins were dominant, while myelin specific proteins were barely detectable in the PNS myelin fraction. At 18 embryonic days, however, BR‐PO, BP and PASII proteins became the main protein components of the PNS myelin, whereas high molecular weight proteins decreased in quantitative importance during development. At the early stage of myelination other glycoproteins were also detectable in the PNS myelin. Radioactive fucose was actively incorporated into the two glycoproteins, BR‐P0 and PASII, at the early stage of myelination in vivo. These results suggested that myelin proteins especially glycoproteins, may play an important role in PNS myelin formation.

Lipid binding activities of the P2 protein in peripheral nerve myelin

Lipid binding activities of the P2 protein in peripheral nerve myelin were examined using retinoic acid, retinol and oleic acid as ligands. The P2 protein showed the specific binding affinity to both of retinoic acid and retinol. The binding site of these ligands was suggested to be similar. In addition, the high binding activity of the P2 protein with oleic acid was also observed. The ligands specificities of the P2 protein are clearly different from those of cellular retinoic acid binding protein (CRABP), cellular retinol binding protein (CRBP), and Z protein. In amino terminal sequence, however, the P2 protein contained considerable homologous structure to these lipid binding proteins. Therefore, the P2 protein and these lipid binding proteins may belong to a family of structurally related proteins evolved from a common ancestral gene.

The Complete Amino Acid Sequence of Human P2 Protein

Abstract: The complete amino acid sequence of P2 protein from human peripheral nerve myelin was determined from nine staphylococcal protease peptides and four cyanogen bromide peptides. Human P2 protein is composed of 131 amino acids and has a molecular weight of 14,818. Compared to bovine P2 protein, there are replacements at nine positions (human↔bovine): 18(Asp↔Glu), 39(Thr↔Arg), 56(Thr↔Pro), 83(Ile↔Thr), 87(Gln↔Ala), 96(Arg↔Lys), 100(Lys↔Asn), 115 (Ala↔Val), and 121(Gly↔Asp).

Neuritogenic Determinant of Bovine P2 Protein in Peripheral Nerve Myelin

Abstract: Experimental allergic neuritis (EAN) is an experimentally produced demyelinating disease of peripheral nervous system. Several peptides of bovine P2 protein were tested for neuritogenic activity in Lewis rats. The hexacosapeptide CiT4 (residues 53‐78 of bovine P2 protein) showed the highest neuritogenic activity among the peptides tested. The nonapeptide (residues 70‐78) and the tridecapeptide (residues 66‐78) were synthesized using the liquid phase peptide synthesis technique. The tridecapeptide showed mild, but definite activity in inducing EAN in the rats, while the nonapeptide was inactive. The localization of the neuritogenic determinant of bovine P2 protein in Lewis rats is discussed.

DISTRIBUTION AND OPTICAL ACTIVITY OF THE BASIC PROTEIN IN BOVINE PERIPHERAL NERVE MYELIN

Abstract— Antiserum to BF protein isolated from bovine spinal roots has been used to study the distribution of the protein in other species and tissues.

On basic proteins in bovine peripheral nerve myelin.

1. Myeline proteins in bovine peripheral nerve migrated as two main band-(BF and BR protein) and one faint middle band (BM protein) on sodium dodecyls sulfate-polyacrylamide gel electrophoresis. The relative mobility of these two main bands differed from those of myelin proteins in the central nervous system. 2. The acid extract of the myelin fraction from bovine peripheral nerve was separated into one main peak and two minor peaks on a Sephadex G-75 column. The major component of the second minor peak was the BM protein; the major component of the main peak was the BF protein. The BR protein was not extractable by acid solution. 3. Molecular weights of the BF, the BM and the BR protein were determined as around 13 000, 20 000 and 28 000, respectively, by sodium dodecylsulfate-polyacrylamide gel electrophoresis. 4. The amino acid composition of the BF protein was quite different from the encephalitogenic protein and the Folch-Lees type proteolipid protein in the central nervous system. However the BM protein showed similar amino acid composition to the encephalitogenic protein. 5. The tryptic peptide maps of the BF protein and of the encephalitogenic protein were quite different. The results suggested that the amino acid sequences of these two proteins are different and that they contain no common tryptophan-containing peptide.

Amino acid sequence of the glycopeptide derived from a major glycoprotein in bovine peripheral nerve myelin

The major protein in peripheral nerve myelin, which is not found in central nerve myelin, is a glycoprotein [ 1,2] . This glycoprotein, which accounts for >5% of total peripheral myelin protein [3] , increases rapidly in parallel to myelination of peripheral nerve at developmental stages [4] and is suggested to localize in interperiod line of myelin lamellae [5] . .The glycoprotein may play important roles in myelin membrane organization or adhesion. One of the difficulties in studying the membrane glycoprotein was its solubilization and subsequent purification. Recently, it became possible to obtain the myelin glycoprotein, referred to as BR protein (mol. wt 28 000), in highly purified form from bovine peripheral myelin by a gel filtration method with SDS solution [6] . The glycoprotein as PO protein was also purified from rabbit sciatic nerve myelin by a similar method [7]. The amino acid composition, carbohydrate composition and N-terminal amino acid of the glycoprotein have been reported [6,7]. This study describes the amino acid sequence of the BR-PO protein in the neighbourhood of a carbohydrate moiety.

Studies on Myelin Proteins in Human Peripheral Nerve

The myelin fraction from human peripheral nerve was prepared. Two basic protein fractions (BF-P2 and PB) were isolated from acid extracts of the myelin fraction and three glycoproteins (BR-PO, PASII and Y protein) were purified from its acid-insoluble residue. In biochemical analysis the human BF-P2 protein (M.W.13,000) showed similar but not identical properties to bovine BF-P2 protein. The PB fraction was suggested to include the encephalitogenic CNS-BP (M.W.18,000) and another, new protein of similar molecular weight. Both the human BR-PO protein (M.W.28,000) and PASII protein (M.W.13,000) showed similar biochemical properties to the corresponding myelin proteins of bovine peripheral nerve, while they both are clearly different from other myelin proteins. Close relationship between the BR-PO protein and the Y protein (M.W.22,000) was suggested by amino acid analysis. Injection of the myelin fraction of bovine peripheral nerve with the complete adjuvant produced EAN while the CNS-BP induced EAE in laboratory animals. However, all three purified proteins, BF-P2, BR-PO and PASII, from bovine peripheral nerve myelin were inactive in inducing demyelinating diseases.