Myelin/oligodendrocyte glycoprotein (MOG) is a primary target autoantigen in experimental autoimmune encephalomyelitis, a widely used animal model for autoimmune demyelinating diseases such as multiple sclerosis. We have isolated several rat MOG cDNAs and confirmed their identity by comparison with MOG N-terminal peptide sequence. As expected, MOG mRNA expression is CNS-specific and peaks during active myelination. Our studies show that full length MOG mRNA is approximately 1.6 kb and encodes a signal peptide of 27 amino acids, followed by 218 residues for mature MOG (24,962 MW). A single site for N-glycosylation is found at Asn-31. Rather than the ubiquitous AAUAAA polyadenylation signal, a series of three overlapping, rare poly A signals were identified. The N-terminal half of mature MOG shares 52% identity with bovine butyrophilin, a possible lipid receptor. This same region has 39% identity with chicken B-G antigen, a major histocompatibility complex antigen involved in B cell selection and immune repertoire development. We show that both MOG and butyrophilin, each exhibiting a single Ig-like variable region domain, meet criteria for inclusion in the immunoglobulin superfamily. Moreover, MOG appears to represent a unique member of this superfamily in that it possesses two potential transmembrane domains, in contrast to a single membrane-spanning domain or glycophospholipid anchor found in all other members of Ig superfamily members.

The myelin/oligodendrocyte glycoprotein (MOG) is found exclusively in the CNS, where it is localized on the surface of myelin and oligodendrocyte cytoplasmic membranes. The monoclonal antibody 8-18C5 identifies MOG. Several studies have shown that anti-MOG antibodies can induce demyelination, thus inferring an important role in myelin stability. In this study, we demonstrate that MOG consists of two polypeptides, with molecular masses of 26 and 28 kDa. This doublet becomes a single 25-kDa band after deglycosylation with trifluoromethanesulfonic acid or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, indicating that there are no or few O-linked sugars and that the doublet band represents differential glycosylation. Partial trypsin cleavage, which also gave a doublet band of lower molecular weight, confirmed this idea. MOG was purified by polyacrylamide gel electrophoresis, followed by electroelution. Three N-terminal sequences of eight to 26 amino acids were obtained. By western blot analysis, no binding was found between MOG and cerebellar soluble lectin. MOG does not seem to belong to the signal-transducing GTP-binding proteins. Reduced MOG concentrations were observed in jimpy and quaking dysmyelinating mutant mice, giving further support to its localization in compact myelin of the CNS.

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The third component of human complement (C3) is a key molecule in the activation of the complement cascade. C3 cDNA fragments were used to identify seven cosmid clones that covered all but 1 kilobase pair (kb) of the C3 gene. The remainder of the gene was cloned by using the polymerase chain reaction. These clones were used to identify the intron/exon boundaries and to map the gene. The C3 gene is 42 kb in length and comprises 41 exons ranging in size from 52 to 213 base pairs (bp). The transcription start site was identified by primer extension, and approximately 1 kb of DNA upstream of this site was sequenced. Putative TATA and CAAT boxes were identified along with a number of regions that shared homology with known regulatory sequences. These include responsive elements for interferon-gamma, interleukin-6, nuclear factor kappa B, estrogen, glucocorticoids and thyroid hormone. Several of these agents have been shown to affect C3 synthesis and mRNA levels. The sizes of the exons in C3 were compared to those of C4 and alpha 2-macroglobulin (alpha 2M). Thirty-nine of 41 exons in C4 were found to be of similar size to the analogous ones in C3, and two-thirds of those in alpha 2M were also similarly sized, supporting the hypothesis that these genes arose from a common ancestor.

The myelin/oligodendrocyte glycoprotein (MOG) is identified by monoclonal antibody 8–18C5. MOG is localized on the surface of myelin and oligodendrocyte processes. Recently, several studies have shown that MOG plays an important role as a target for antibody-induced demyelination. In the present study, we investigated MOG expression in the brains of normal and myelin-deficient (mld)mutant mice during development. By gel electrophoresis and immunoblotting, we observed the developmental pattern of two closely migrating bands, with apparent molecular masses of 26 and 28 kilodaltons. Their concentrations increased coordinately during the most active phase of myelin and myelin basic protein (MBP) synthesis. Between 20 and 25 days of age, the MOG developmental pattern superimposed that of MBP as well as myelin yields. In mld mutant mice, which are affected by a severe deficit of MBP synthesis, MOG was present at reduced levels (40% of controls at 60 days of age). At 85 days of age, mld mice exhibited increased concentrations of MBP, and myelin was better compacted. At this age, MOG concentrations increased and reached 70% of controls. These results suggest that MOG could play a role in the maintenance or completion of the myelin sheath. Its expression level may be modulated by the presence of compact myelin and/or MBP in the myelin sheath.

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The membrane-restricted photoactivatable carbene generator 3-(trifluoromethyl)-3-(m-[125I]-iodophenyl)diazirine [Brunner, J., & Semenza, G. (1981) Biochemistry 20, 7174-7182] was used to label the subunits of the membrane attack complex of complement (C5b-9). C5b-9 complexes either were assembled from serum on erythrocyte membranes or were reconstituted from purified components on liposomes. After irradiation, most of the probe is bound to C9 independent of the membrane system used, indicating that the wall of the transmembrane channel is predominantly composed of C9. No difference was observed whether polymerized C9 was in the tubular or nontubular form [Podack, E. R., & Tschopp, J. (1983) J. Biol. Chem. 257, 15204-15212], showing that tubule closure is not essential for successful lipid insertion. The same label distribution between the two forms of polymerized C9 was obtained by analyzing zinc-polymerized C9 in the absence of C5b-8. Since the photoreactive probe reacted with at least two distinct polypeptide segments within C9, lipid interaction does not occur via a single segment of hydrophobic amino acids.

p36 is a major substrate of both viral and growth factor receptor associated protein kinases. This protein has recently been named calpactin I heavy chain since it is the large subunit of a Ca2(+)-dependent phospholipid and actin binding heterotetramer. The primary structure of p36 has been determined from analysis of cloned cDNA. The protein contains 338 amino acids, has an approximate molecular weight of 39,000, and is comprised of several distinct domains, including four 75 amino acid repeats. From two overlapping cosmid clones isolated from different mouse genomic liver libraries, the complete intron/exon structure of the p36 gene was determined and the 5' and 3' noncoding regions of the gene were analyzed. The coding and 3' untranslated region of the p36 gene contains 12 exons which range in size from 48 to 322 base pairs (bp) with an average size of 107 bp. The repeat structures found at the protein level are not delineated by single exons, but the N-terminal p11-binding domain is encoded by a single exon. Structural mapping of the gene demonstrated that the lengths of the first two introns in the coding region are together approximately 6 kilobases (kb), while the other introns range in size from 600 to 3600 bp with an average size of 1650 bp. The p36 gene is at least 22 kb in length and has a coding sequence of approximately 1 kb, representing only 4.5% of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)