Sequencing and cloning of the cDNA of guinea pig eosinophil major basic protein.

Major basic protein (MBP) purified from guinea pig eosinophils elicited histamine release from rat peritoneal mast cells at concentrations higher than 3 micrograms/ml both in the presence and in the absence of extracellular Ca2+. After reverse-phase high-performance liquid chromatography, it was revealed that MBP was composed of two different proteins with quite similar molecular weights and pI values, although the amino acid compositions were slightly different. The partial amino acid sequence of one of these MBPs was determined and the primers for the polymerase chain reaction (PCR) were synthesized according to the partial amino acid sequence. Using these primers and the cDNAs obtained from guinea pig eosinophils, the PCR was carried out in order to synthesize the hybridization probe of MBP for screening the cDNA library. After screening with 8 x 10(5) clones, a positive clone, which encoded a full length of pre-proMBP, was obtained. According to the sequencing data of this clone, it was revealed that pre-proMBP was composed of 3 domains; signal peptide, acidic domain and mature MBP. The predicted pI value of mature MBP was 11.7, though that of proMBP was 7.8. The homology in the amino acid sequence between guinea pig proMBP and human proMBP was 49.4%, while guinea pig mature MBP was more homologous (58%) to human mature MBP.     

Characterization of Basic Proteins from Goldfish Myelin

Abstract: Myelin basic protein (MBP) from common goldfish ( Carassius auratus ) myelin was extracted with dilute mineral acid. Immunological cross-reactivity of the goldfish MBP, with polyclonal antisera raised against bovine MBP, suggested that the goldfish protein has epitopes for these antibodies. It also reacted with a monoclonal antibody specific for a seven amino acid epitope (130–137) conserved in the MBP of most mammalian species. To characterize the charge heterogeneity of this protein, we iodinated the protein with ¹²⁵I and chromatographed it on a carboxymethyl cellulose-52 column together with a nonlabeled acid soluble fraction prepared from human white matter as a carrier protein. All of the goldfish protein was recovered in the unbound fraction, demonstrating that it was less cationic than the carrier protein (human MBP). We have also examined the urea alkaline gel profile of the goldfish MBP together with the human C-1, C-2, C-3, C-4, and C-8 components. The results from these experiments indicated that this MBP extracted from goldfish brain myelin lacked the microhet-erogeneity that is associated with MBPs from higher vertebrates. The MBPs from goldfish myelin were separated into their isoforms by reversed-phase HPLC. Amino acid compositions were determined for both the 17- and 14-kDa goldfish proteins. Amino acid analysis revealed similarities with the compositions of other MBPs; however, the serine content in both the 17- and 14-kDa proteins was higher than that of the human C-1, the mouse C-1 protein, and the shark proteins. The HPLC-purified 14-kDa goldfish protein was chemically cleaved with CNBr for partial sequence analysis. Even from the limited sequence obtained, the sequence ATAST was found in goldfish, which is also present in human, rabbit, and guinea pig MBPs.     

Shark Myelin Basic Protein: Amino Acid Sequence, Secondary Structure, and Self-Association

Myelin basic protein (MBP) from the Whaler shark (Carcharhinus obscurus) has been purified from acid extracts of a chloroform/methanol pellet from whole brains. The amino acid sequence of the majority of the protein has been determined and compared with the sequences of other MBPs. The shark protein has only 44% homology with the bovine protein, but, in common with other MBPs, it has basic residues distributed throughout the sequence and no extensive segments that are predicted to have an ordered secondary structure in solution. Shark MBP lacks the triproline sequence previously postulated to form a hairpin bend in the molecule. The region containing the putative consensus sequence for encephalitogenicity in the guinea pig contains several substitutions, thus accounting for the lack of activity of the shark protein. Studies of the secondary structure and self-association have shown that shark MBP possesses solution properties similar to those of the bovine protein, despite the extensive differences in primary structure.     

In vivo phosphorylation of myelin basic proteins: single and double isotope incorporation in developmentally related myelin fractions

The phosphorylation of myelin basic proteins (MBPs) was studied in developing mouse brain. Based on our previous work we postulated that phosphorylation of MBPs takes place prior to their appearance in the myelin compartment as well as within the myelin sheath. To further test this hypothesis we utilized a subfractionation protocol that yields brain fractions enriched in myelin membranes of differing developmental stages. Incorporation of radioactive phosphate into MBPs was studied in each of the subcellular fractions. After 5- and 15-min incubations of isotope in vivo the highest specific radioactivities (SAs) of MBPs were found in the least mature myelin fractions. Incorporation of 32P in MBPs was greater into serine residues than threonine residues in all of the subcellular fractions studied. The relative turnover of MBP phosphates was studied in each of the subcellular myelin fractions using a time-staggered, double isotope methodology. The most rapid equilibration of MBP phosphates with the trichloroacetic acid (TCA)-soluble phosphate pool occurred in the most mature myelin fractions indicating that the highest turnover of MBP phosphates occurs in the most mature myelin fractions. The SAs and turnover rates of each of the four commonly observed mouse MBPs (14, 17, 18.5, and 21.5 kDa) were similar in any particular subfraction demonstrating that the MBP phosphotransferase system(s) acts on each of the MBPs in a similar manner.     

Purification and structure of caltrin-like proteins from seminal vesicle of the guinea pig

Two different small proteins that cross-react with the antiserum against bovine caltrin (calcium transport inhibitor) have been purified from the seminal vesicle contents of the guinea pig. The primary structure and some molecular characteristics of the pure proteins are reported. The two proteins interact with concanavalin A indicating the presence of carbohydrates in their molecules. Chemical deglycosylation with trifluoromethanesulfonic acid, after reduction and carboxymethylation, results in complete loss of affinity for the lectin. Removal of sugar components from the structure destroys the ability of caltrin-like proteins to react with antibodies to bovine caltrin. The protein moving faster on polyacrylamide gel electrophoresis is designated guinea pig caltrin I, the other is II. They contain 45 and 55 amino acids, and the molecular weights of the peptide portions are 5082 and 6255, respectively. Although they have entirely different amino acid sequences, they share some common features: recognition by rabbit antibodies to bovine caltrin, the predominance of basic residues and the presence of 3 cysteine residues in fraction I and 8 in fraction II. The proteins have pI values of 9.5 and 10.2, respectively, which are consistent with the amino acid composition. The two pure fractions are approximately equally effective, on a weight basis, as inhibitors of 45Ca2+ uptake by guinea pig spermatozoa. The data presented reinforce the hypothesis that caltrin-like proteins are responsible for the previously reported (Coronel, C.E., San Agustin, J., and Lardy, H.A. (1988) Biol. Reprod. 38, 713-722), calcium-transport inhibitor activity detected in reproductive tract fluid from adult male guinea pigs.     

Isolation and characterization of mannan-binding proteins from chicken liver

Two binding proteins which recognize and bind mannose and N-acetylglucosamine (mannan-binding proteins, MBP) have been isolated from chicken liver to near homogeneity mainly by affinity chromatography on a column of Sepharose 4B-mannan. The neutral binding protein (pI 7.0), which has a high glycine content, is an analog of mammalian liver MBP (F-I). F-I consists of a series of proteins composed of two subunits of 28,000 (A) and 32,000 (B) Da. The proteins have molecular weights ranging from 280,000 to 740,000 and subunit compositions ranging from 6A + 4B to 5A + 19B. With increasing molecular weight the specific activity of mannan binding increases gradually, accompanied by a slight change in specificity to a preference for mannose rather than N-acetylglucosamine. The acidic binding protein (pI 5.1) is a glycoprotein with a high glutamic acid content (F-II). The molecular weight of F-II was estimated to be 640,000, and it is composed of single subunits of 41,000 Da. The two MBPs isolated in this study are distinct from the liver lectin specific for N-acetylglucosamine-terminated glycoproteins isolated from the same source [T. Kawasaki and G. Ashwell (1977) J. Biol. Chem. 252, 6536-6543] in chemical properties and binding specificities.     

ADP-ribosylation of myelin basic protein by cholera toxin.

Cholera toxin ADP-ribosylates four types of myelin basic proteins (MBPs) of Mr 14,000, 17,500, 19,000 and 22,000 in rat brain myelin. On an analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, MBP underwent mono- and multi-(ADP-ribosyl)ation by cholera toxin and thus modified MBP migrated on the gel as several discrete protein bands, the molecular masses of which were apparently larger by 500-2000 daltons than that of the corresponding untreated MBP. On average, 1.1 mol of ADP-ribosyl residue was incorporated into 1 mol of MBP. Four types of purified MBPs were also ADP-ribosylated by cholera toxin dependent on GTP and the protein factor for the ADP-ribosylation. The results show evidence that MBP is one of major and specific substrates of cholera toxin in brain membranes.     

Developmental Study of Myelin Basic Protein Variants in Various Regions of Pig Nervous System

A developmental study of myelin basic protein (MBP) variants in eight regions of pig nervous system (NS) was performed using a quantitative electroimmunoblotting procedure. Four major MBP forms with apparent molecular weights of 21.5K, 20.2K, 18.5K, and 17.3K were identified in both the CNS and the PNS and were detected as early as 22 days before birth. Quantification of the most abundant forms, the 21.5K and 18.5K MBPs, revealed characteristic profiles of accumulation of these two variants in different regions of the NS. The ratio of 21.5K:18.5K MBP varied with developmental time as well as with the various NS regions, peaking 20 days postnatally. The 17.3K MBP was observed from embryonic stages to adulthood, as were the 21.5K and 18.5K forms. In contrast, the 20.2K variant appeared most abundant from 10 days before to 22 days after birth and thereafter decreased in intensity so as to be no longer detectable in the brain of a 5-year-old pig. A similar pattern was also observed with an anti-MBP-reacting protein with an apparent molecular weight of 23K. Taken together, these results suggest that in the pig NS, the expression of MBP variants may be regulated both regionally and developmentally.     

Antipeptide antibodies reveal interrelationships of MBP 200 and MBP 235: unique apoB-specific receptors for triglyceride-rich lipoproteins on human monocyte-macrophages

Two human monocyte-macrophage (HMM) membrane binding proteins, (MBP) 200 and 235, are receptor candidates that bind to the apolipoprotein (apo)B-48 domain in triglyceride-rich lipoproteins for uptake independent of apoE. Microsequence analysis of the purified reduced MBP 200R characterized tryptic peptides of MBP 200R. A synthetic peptide mimicking a unique, unambiguous 10-residue sequence (AEGLMVTGGR) induced antipeptide antibodies that specifically recognized MBP 200, 235 and 200R, in 1- and 2-dimensional analyses, indicating 1) the ligand binding protein was sequenced and 2) MBP 200 and 235 yielded MBP 200R upon reduction. These antibodies identified the MBPs in human blood-borne, THP-1, U937 MMs, and endothelial cells (EC) but not in human fibroblasts or Chinese hamster ovary (CHO) cells. Fluorescence activated cell sorting (FACS) analysis located the MBPs on the MM surface as necessary for receptor function. The 10-residue, unambiguous MBP 200-derived sequence is unique, with no matches in extant protein databases. Antipeptide antibodies bind to the MBPs in reticuloendothelial cells that have this receptor activity, but not to proteins in cells that lack this receptor activity. These studies provide the first direct protein sequence and immunochemical data that a new, unique apoB receptor for triglyceride-rich lipoproteins exists in human monocytes, macrophages, and endothelial cells.     

Myelin basic protein is affected by reduced synthesis of myelin proteolipid protein in the jimpy mouse.

Myelin basic proteins (MBPs) from 6-day-old, 10-day-old, 20-day-old and adult normal mouse brain were compared with those from 20-day-old jimpy (dysmyelinating mutant) mouse brain to determine the effect of reduced levels of proteolipid protein (PLP) on MBPs. Alkaline-urea-gel electrophoresis showed that 6-day-old and 10-day-old normal and jimpy MBPs lacked charge microheterogeneity, since C8 (the least cationic of the components; not be confused with complement component C8) was the only charge isomer present. In contrast, MBPs from 20-day-old and adult normal mouse brain displayed extensive charge microheterogeneity, having at least eight components. A 32 kDa MBP was the major isoform observed on immunoblots of acid-soluble protein from 6-day-old and 10-day-old normal and 20-day-old jimpy mouse brain. There were eight bands present in 20-day-old and adult normal mouse brain. Purified human MBP charge heteromers C1, C2, C3 and C4 reacted strongly with rat 14 kDa MBP antiserum, whereas the reaction with human C8 was weak. This suggested that MBPs from early-myelinating and jimpy mice did not react to MBP antisera because C8 was the major charge isomer in these animals. Purification of MBPs from normal and jimpy brain by alkaline-gel electrophoresis showed that both normal and jimpy MBPs have size heterogeneity when subjected to SDS/PAGE. However, the size isoforms in normal mouse brain (32, 21, 18.5, 17 and 14 kDa) differed from those in jimpy brain (32, 21, 20, 17, 15 and 14 kDa) in both size and relative amounts. Amino acid analyses of MBPs from jimpy brain showed an increase in glutamic acid, alanine and ornithine, and a decrease in histidine, arginine and proline. The changes in glutamic acid, ornithine and arginine are characteristic of the differences observed in human C8 when compared with C1.     

In Vitro Synthesis of the Myelin Basic Proteins: Subcellular Site of Synthesis

Abstract: Brains of 3-week-old C57BL/6J mice were homogenized and fractionated into several subcellular components, each of which was examined for ability to synthesize the myelin basic proteins (MBPs) in vitro. Myelin basic proteins were purified from incubation mixtures by conventional means. That the products of synthesis were the myelin basic proteins was established by solubility at pH 3, co-chromatography with authentic proteins on carboxymethylcellulose and co-migration with standards in two different polyacrylamide gel electrophoretic systems. The fractions examined for their ability to synthesize MBPs were the whole homogenate, postnuclear supernatant, postmitochondrial supernatant, crude mitochondrial pellet, free ribosomes and bound ribosomes. Although there was no requirement for exogenous energy sources for protein synthesis in the whole homogenate, as the homogenate was fractionated an increasing requirement emerged. Most of the label in the MBP preparations from whole homogenate and postnuclear supernatant incubations migrated with the large (L) and small (S) MBPs on gel electrophoresis; however, as the homogenate was subfractionated and incubated, a greater percentage of the label migrated more slowly than L and S on acetic acid-urea gels. To show synthesis of the MBPs the L and S bands were cut out of these gels and rerun on sodium dodecylsulfate gels. Alternatively, MBP preparations were subjected directly to two-dimensional gel electrophoresis and the bands corresponding to L and S were excised and counted. With this method only the whole homogenate, postnuclear supernatant, postmitochondrial supernatant and free ribosomes were observed to synthesize the MBPs in vitro. The "bound" ribosomes were not observed to synthesize significant amounts of the MBPs, incubated either intact or released from the membrane. It was concluded that the free ribosomes are the principal site of synthesis of the myelin basic proteins in the brain.     

A simple and rapid method for isolating myelin basic protein

The conduction of impulses along axons of nerves is facilitated by the myelin sheath, composed of proteins and lipid. Myelin basic proteins (MBPs) are extrinsic membrane proteins that play an important role in the structural organization of the myelin sheath. In the central nervous system, MBPs account for 30-40% of total protein. The traditional method of MBP isolation involves the use of chloroform-ethanol, which would destroy the native form of MBP. A modified method for maintaining its native form was developed. The white matter of porcine brain was directly extracted by buffers containing different concentrations of sodium chloride owing to MBP solubilized at high concentration of NaCl. The MBP was further purified by cation exchange chromatography and buffers containing glycine and salts. Purified MBP were consistently obtained by this method.     

Synthesis and incorporation of myelin polypeptides into CNS myelin

The distribution of newly synthesized proteolipid protein (PLP, 23 kdaltons) and myelin basic proteins (MBPs, 14-21.5 kdaltons) was determined in microsomal and myelin fractions prepared from the brainstems o1 10-30 d-old rats sacrificed at different times after an intracranial injection of 35S-methionine. Labeled MBPs were found in the myelin fraction 2 min after the injection, whereas PLP appeared first in the rough microsomal fraction and only after a lag of 30 min in the myelin fraction. Cell-free translation experiments using purified mRNAs demonstrated that PLP and MBPs are synthesized in bound and free polysomes, respectively. A mechanism involving the cotranslational insertion into the ER membrane and subsequent passage of the polypeptides through the Golgi apparatus is consistent with the lag observed in the appearance of the in vivo-labeled PLP in the myelin membrane. Newly synthesized PLP and MBPs are not proteolytically processed, because the primary translation products synthesized in vitro had the same electrophoretic mobility and N-terminal amino acid sequence as the mature PLP and MBP polypeptides. It was found that crude myelin fractions are highly enriched in mRNAs coding for the MBPs but not in mRNA coding for PLP. This suggests that whereas the bound polysomes synthesizing PLP are largely confined to the cell body, free polysomes synthesizing MBPs are concentrated in oligodendrocyte processes involved in myelination, which explains the immediate incorporation of MBPs into the developing myelin sheath.     

An Approach to Searching Protein Sequences for Superfamily Relationships or Chance Similarities Relevant to the Molecular Mimicry Hypothesis: Application to the Basic Proteins of Myelin

A rapid method for similarity searches (FASTP program) was used to identify similarities between a protein database and the human basic proteins from myelin [P2 protein and 17.2K, 18.5K, and 21.5K variants of myelin basic protein (MBP)]. From similarity scores, we concluded that none of the presently known proteins are in a family containing the MBPs. No new members were found for the lipid-binding family of which P2 is a member. Sequence similarities deemed relevant to the molecular mimicry hypothesis for virus-induced autoimmunity were identified in FASTP data with the aid of microcomputer programs. Several MBP/viral protein similarities were found that have not been reported previously. Of note because of their association with demyelinating conditions were proteins from visna and vaccinia. Similarity with visna was specific to the 21.5K and 20.2K MBPs. The most interesting new possibility for mimicry involving the P2 protein was between the influenza A NS2 protein and a sequence region of P2 thought to be neuritogenic in animals and mitogenic for lymphocytes from some patients with Guillain-Barré syndrome (GBS). This may have relevance for some cases of GBS associated with the 1976 U.S.A. swine flu vaccination program. Because FASTP reports only the best similarities between proteins, searches with FASTP may not have detected all the examples of mimicry present in the database. Searches might also be more effective if similarities could be scored on immunological rather than structural relatedness.     

Immunochemical cross-reactivity between intact purified myelin basic protein (MBP) and the synthetic encephalitogenic peptide S49

Three antisera to myelin basic protein—a rabbit antiserum pool against rat myelin, a rabbit antiserum pool against rat myelin basic protein (MBP), and a monkey antiserum against bovine MBP—were found to contain detectable levels of antibodies that would bind radiolabeled S49 (GSLPQKAQRPQDENG). Strongly encephalitogenic in Lewis rat, S49 is a synthetic peptide representing residues 69–84 of bovine MBP with a deletion of glycine-76 and histidine-77 to make it analogous to rat and guinea pig MBPs. The rabbit antimyelin antiserum and the monkey anti-MBP antiserum contained antibodies directed against a non-sequential determinant that required asparagine 84, the glycine-histidine deletion, and residues 69–71 for maximal activity. S49-reactive antibodies from the rabbit anti-MBP antiserum were directed solely against a sequential determinant comprising residues 69–71. S49-reactive antibodies from all three antisera reacted in liquid phase with purified intact rat, guinea pig, and bovine MBP showing that the determinant is exposed for B cell recognition even in bovine MBP and can serve both as immunogen and reactant.This work supported at Duke University Medical Center by Research Grant NS-10237 from the National Institutes of Health of the U.S. Public Health Service and the Medical Scientist Training Program Grant #5-T32-OMO-7171-08; at St. Luke's Hospital Center by NS-15322 from the National Institutes of Health of the U.S. Public Health Service; and at Northwestern University by Research Grant NS-06262 from the National Institutes of Health of the U.S. Public Health Service.     

Epitopes of peptide 43-88 of guinea pig myelin basic protein: localization with monoclonal antibodies

Two monoclonal antibodies, one raised by immunization with mouse myelin basic protein (MBP) and the second raised by immunization with peptide 68-88 of guinea pig MBP, were compared with respect to specificity. The former antibody (15.32) cross-reacted completely with rat, guinea pig, human, and bovine MBP. It also reacted with peptide 43-88 from each MBP. The latter antibody (22.17) was nonreactive with MBP, but cross-reacted with peptide 43-88 from rat, human, guinea pig, and bovine MBP. When tested with small peptides derived from peptide 43-88, antibody 22.17 reacted with an epitope in the C-terminal region. Antibody 15.32 reacted with an epitope in the N-terminal half of the peptide. The data show that 22.17 reacted with a unique epitope associated only with free peptide, whereas 15.32 recognized an epitope common to both peptide 43-88 and MBP.     

The ectopic expression of myelin basic protein isoforms in Shiverer oligodendrocytes: implications for myelinogenesis

The myelin basic proteins (MBPs) are a set of peripheral membrane polypeptides that are required for the compaction of the major dense line of central nervous system myelin. We have used primary cultures of oligodendrocytes from MBP-deficient shiverer mice as host cells for the expression by cDNA transfection of each of the four major MBP isoforms. The distributions of the encoded polypeptides were studied by immunofluorescence and confocal microscopy and compared with patterns of MBP expression in normal mouse oligodendrocytes in situ and in culture. The exon II-containing 21.5- or 17-kD MBPs were distributed diffusely in the cytoplasm and in the nucleus of the transfectants, closely resembling the patterns obtained in myelinating oligodendrocytes in 9-d-old normal mouse brains. By contrast, the distribution of the 14- and 18.5-kD MBPs in the transfectants was confined to the plasma membrane and mimicked the distribution of MBP in cultures of normal adult oligodendrocytes. Our results strongly suggest that the exon II-containing MBPs are expressed first and exclusively during oligodendrocyte maturation, where they may play a role in the early phase of implementation of the myelination program. In contrast, the 14- and 18.5-kD MBPs that possess strong affinity for the plasma membrane are likely to be the principle inducers of myelin compaction at the major dense line.