Sites in Myelin Basic Protein that React with Monoclonal Antibodies

The epitopes (antigenic sites) for seven monoclonal antibodies (MAbs) evoked in rats or mice by guinea pig or monkey myelin basic protein (BP) have been located in four different sequences of the BPs extracted from various species. Six of the MAbs were evoked by guinea pig BP. (1) One epitope, possibly a pair, is included within residues 1-14 of all BPs tested and reacts with two rat IgG MAbs. (2) A definite pair of overlapping epitopes includes the central Phe91-Phe92 sequence. One epitope is contained entirely within sequence 90-99 and reacts with a rat IgG MAb. The substitution of Ser in chicken BP for Thr97 destroys this epitope. The other epitope appears to include residues on the amino side of Phe44 and even of His32 and suggests some tertiary structure in BP. This epitope reacts with a mouse IgM MAb that does not recognize the chicken substitution. (3) The third epitope lies within residues 114-121, specifically including Trp118, and reacts with a rat IgG MAb. A cross-reacting epitope probably includes residues 44-45 in certain species (guinea pig and bovine but not rabbit). (4) Another pair of epitopes is located within residues 131-140 but is severely species-restricted. This region in guinea pig BP evoked a species-specific mouse IgM MAb. The same region in monkey BP evoked the seventh MAb, a mouse IgG, which reacts with human, chimpanzee, monkey, bovine, and rat-18.5 kDa BPs and to a lesser extent rabbit BP but not with guinea pig, pig, or chicken BPs. Some tertiary structure in guinea pig BP is also suggested by the reactivities with the IgM MAb. All of the MAbs react with myelin in histologic preparations, but the optimum method of preparation of the tissue varies with each.     

Cloning of the cDNA encoding neural adhesion molecule F3 from bovine brain

We cloned a bovine cDNA encoding the neural adhesion molecule F3 and analyzed its nucleotide sequence. The coding region consisted of 3054 bp encoding 1018 amino acid (aa) residues. The M_r calculated from the deduced as sequence was 113 383. Bovine F3 had 93, 94 and 77% as identity with the mouse, human and chicken homologs, respectively. Bovine F3, similar to those of chicken and human, was devoid of two as residues (Ile-Thr) in the sixth immunoglobulin type C2-like domain, as compared with the mouse homolog. Parts of bovine F3 protein were overproduced in Escherichia coli. The antibodies raised against the recombinant proteins in rabbits reacted specifically with F3. F3 protein was detected in cerebellum, cerebrum and spinal cord in Western blot analysis.     

Primary structure of the amino-terminal (vitamin K-dependent) region of human prothrombin

The amino acid sequence of residues 1–51 of human prothrombin fragment 1 has been determined. This 51 residue peptide is 1 residue shorter than the comparable bovine region and 8 of the amino acid residues are different. The positions of 10 glutamic acid residues are identical to the γ-carboxylated ones in the bovine species. From this homology and additional evidence, these residues in human prothrombin are considered to be γ-carboxylated. The sequence was completed by automated Edman degradation of the reduced, alkylated fragment and its subfragmentation with trypsin, thermolysin and acid hydrolysis.     

Paramyxovirus membrane protein enhances antibody production to new antigenic determinants in the actin molecule: a model for virus-induced autoimmunity.

Paramyxovirus membrane (M) protein specifically binds to cellular actin but not to bovine serum albumin or myoglobin, as determined by affinity chromatography and enzyme-linked immunosorbent assay. The binding site for M protein on actin is different from the binding sites for antiactin antibodies. The interaction of M protein with actin resulted in production of antibodies to several new antigenic sites on the actin molecule. Five rabbits immunized with actin alone produced antibodies against the N-terminal sequence (residues 1 to 39). Another five rabbits immunized with a mixture of M protein and actin produced antibodies against a C-terminal fragment and a central region as well as the N-terminal fragment. By immunoblotting with proteolytic fragments of actin, the new antigenic sites were located between amino acid residues 40 to 113, 114 to 226, and 227 to 375. Antisera taken from some patients with recent measles virus infections demonstrated antiactin antibodies to sites other than the N-terminal fragment of actin (amino acids 1 to 39). The interaction of paramyxovirus M protein with actin and the subsequent production of antibodies to new antigenic sites may serve as a model for one of the mechanisms of virus-induced autoimmunity.     

Isolation and partial structural characterization of an equine fibrinogen CNBr fragment that exhibits immunologic cross-reactivity with an A alpha-chain cross-linking region of human fibrinogen

Immunochemical studies of equine fibrinogen were conducted to characterize the structural basis for the immunologic cross-reactivity observed between human and equine A alpha chains when employing an antiserum to the 26K, human cyanogen bromide (CNBr) fragment, A alpha 241-476 (CNBr VIII). A 38K, equine CNBr fragment that reacts with this antiserum was isolated from CNBr-digested equine fibrinogen by Sephadex G-100 gel filtration. It was further purified by sequential hydrophobic chromatography on phenyl-Sepharose CL-4B, followed by reversed-phased (C-8) high-performance liquid chromatography (HPLC). NH2-Terminal analysis of the purified fragment, designated EqA alpha CNBr, identified one major sequence whose first three residues, E-L-E, were identical with those of human CNBr VIII. Tryptic and staphylococcal protease digests of the equine fragment were resolved by reversed-phase HPLC (C-4, C-18), and the separated components were characterized by amino acid analysis and automated Edman degradation. A total of 34 tryptic and 20 staph protease peptides yielded sequence information that permitted the alignment of 271 equine residues with residues A alpha 241-517 from the COOH-terminal two-thirds of the human A alpha chain so that 63% of the possible matches were identical. Other features of interest included (1) an amino acid substitution in which the methionine residue at A alpha 476 in the human A alpha chain was replaced by a valine residue, thus accounting, in part, for the larger EqA alpha CNBr fragment obtained from the equine molecule, and (2) a region of striking homology in which 36 successive residues, corresponding to A alpha 428-464 in the human A alpha chain, were identical in both species. These findings, together with available structural data for the COOH-terminal portion of the rat and bovine A alpha chains, indicate that the region corresponding to (human) A alpha 240-517 represents a conserved portion of the fibrinogen molecule. This may, in turn, explain the difficulties encountered when trying to raise monoclonal antibodies to cross-linking regions that are contained within the COOH-terminal two-thirds of the human A alpha chain.     

Immunochemical localization of the C-terminal hexapeptide of histone H3 at the surface of chromatin subunits.

The C-terminal hexapeptide of histone H3 of chicken erythrocytes (residues 130-135) corresponding to the sequence Ile-Arg-Gly-Glu-Arg-Ala ( IRGERA ) was prepared by solid-phase peptide synthesis and, after coupling to bovine serum albumin, was used to elicit antibodies in rabbits. The antigenic activity of the synthetic peptide IRGERA was found to be very similar to that of the natural CN3 fragment (residues 121-135), and it inhibited the H3-anti H3 reaction in complement fixation, solid-phase radioimmunoassay, and enzyme-linked immunosorbent assay. Antibodies induced by IRGERA were found to bind equally well to IRGERA coupled to hemocyanin, to the intact H3 molecule, and to chromatin subunits (nucleosomes and core particles). The results demonstrate that the C-terminal hexapeptide of histone H3 is located at the surface of chromatin subunits and agree with current models proposed for the spatial organization of the chromatin core particle.     

A local antigenic determinant distribution in a continuous antigenic region at residues 38-54 of hen egg white lysozyme

The precise location of the antigenic determinants in a continuous antigenic region at residues 38–54 of hen egg white lysozyme (lysozyme) was investigated using the inhibition test of binding of N^α-[¹⁴C]acetyl fragment 38–54 with goat (three individuals) and sheep (four individuals) anti-lysozyme antisera by various synthetic and proteolytic fragments of lysozyme. From these inhibition studies, we found that in this region there were three independent antigenic determinants, consisting of residues 38–45, 40–48, and 44–54, respectively. The existence and the specificity of the antibodies directed to these determinants were further examined with isolating the specific antibodies by affinity chromatography on columns to which the fragment 38–45, 44–48, and 46–54 were bound. The results indicated that these determinants partially overlapped one another in amino acid sequence, but the antibodies directed to them could recognize only each corresponding determinant. These antibodies were also shown to be reactive with native lysozyme as well as a reduced and S-carboxymethylated derivative of lysozyme, and to be found in goat and sheep anti-lysozyme antibodies. The amounts of these antibodies calculated from the binding capacities were in the range from 0 to 48 μg/ml of antisera. These values corresponded to a small fraction of the total precipitable anti-lysozyme antibodies and were as high as 0.8% of the total. The ratios of the amounts of these antibodies differ in individuals or in different species of animals. The binding affinities of N^α-[¹⁴C]acetyl fragment 38–54 with these antibodies were in the range from 1.3 × 10⁷ to 2.6 × 10⁸m^?1. The double-reciprocal plots of the antigen binding with these antibodies drew almost a straight line compared with those of a mixture of several antibody populations, that is, whole antisera.     

Identification of residues in the CH2/CH3 domain interface of IgA essential for interaction with the human fcalpha receptor (FcalphaR) CD89

Cellular receptors for IgA (FcalphaR) mediate important protective functions. An extensive panel of site-directed mutant IgAs was used to identify IgA residues critical for FcalphaR (CD89) binding and triggering. Although a tailpiece-deleted IgA1 was able to bind and trigger CD89, antibodies featuring CH3 domain exchanges between human IgA1 and IgG1 could not, indicating that both domains but not the tailpiece are required for FcalphaR recognition. To further investigate the role of the interdomain region, numerous IgA1s, each with a point substitution in either of two interdomain loops (Leu-257-Gly-259 in Calpha2; Pro-440-Phe-443 in Calpha3), were generated. With only one exception (G259R), substitutions produced either ablation (L257R, P440A, A442R, F443R) or marked reduction (P440R) in CD89 binding and triggering. Further support for involvement of these interdomain loops was provided by interspecies comparisons of IgA. Thus a human IgA1 mutant, LA441-442MN, which mimicked the mouse IgA loop sequence through substitution of two adjacent residues in the Calpha3 loop, was found, like mouse IgA, not to bind CD89. In contrast, bovine IgA1, identical to human IgA1 within these interdomain loops despite numerous differences elsewhere in the Fc region, did bind CD89. We have thus identified motifs in the interdomain region of IgA Fc critical for FcalphaR binding and triggering, significantly enhancing present understanding of the molecular basis of the IgA-FcalphaR interaction.     

鸡含锰超氧化物歧化酶cDNA克隆及序列分析

 为弄清鸡含锰超氧化物歧化酶 (manganese containingsuperoxidedismutase ,MnSOD)的cDNA序列 ,以开展动物锰营养学的深入研究 ,根据已知鸡MnSOD的N端氨基酸序列设计简并引物 ,应用 3′RACE(rapidamplificationofcDNAends)技术 ,扩增克隆了鸡心肌MnSOD 990bp的 3′cDNA片段 .再根据 3′RACE片段测序结果设计引物进行 5′RACE ,结果获取了一个与 3′RACE片段相互重叠的鸡心肌MnSOD 52 1bp的 5′RACE片段 ,并对其进行了克隆测序 .最后根据 3′RACE片段和 5′RACE片段序列信息进行拼接 ,从而获取鸡MnSODcDNA的全序列信息 .研究结果表明 :鸡MnSODcDNA全长为 110 8个核苷酸 ,其中 5′非翻译区 2 5个核苷酸 ,编码区 675个核苷酸 ,3′非翻译区 4 0 8个核苷酸 ,编码一个长 2 2 4个氨基酸残基的蛋白质前体 .其中信号肽长 2 6个氨基酸残基 ,成熟肽长 198个氨基酸残基 ,分子量为 2 2kD .与人、大鼠、线虫、果蝇等真核生物MnSOD氨基酸序列的同源性分别为82 4 %、84 .7%、62 .4 %、59.3% .     

Internal quadruplication in the structure of human interstitial retinol-binding protein deduced from its cloned cDNA

Interstitial retinol-binding protein (IRBP) is a glycoprotein that shuttles retinoids between the retina and pigment epithelium and is secreted by the photoreceptor cells of the vertebrate eye. Human retina cDNA libraries in lambda gt10 were screened with a previously isolated human IRBP probe (H.4 IRBP), yielding five overlapping cDNA clones generating a 4223-base sequence. A 17-kilobase pair clone (HGL.3) isolated by screening a human genomic library in EMBL3 with H.4 IRBP yielded a 2.5-kilobase pair SstI fragment that overlapped the 5' end of the cDNA sequence by 329 nucleotide residues. An open reading frame encoded the N-terminal sequence of human IRBP and predicted a protein consisting of 1262 amino acids with a molecular mass of 136,600. Two putative N-linked glycosylation sites were identified. The translated sequence suggests that there is a 16-amino acid presumptive signal peptide rich in hydrophobic residues and with a high alpha-helix probability preceding the N terminus of the mature protein. The amino acid sequence of human IRBP could be aligned with 87% identity with the amino acid sequences of 31 peptides (605 residues) purified from a tryptic digest of bovine IRBP. The protein sequence of human IRBP contains four duplicated segments (302-310 residues in length) with 33-38% identity. From the degree of identity between the bovine and human sequences, it is possible that IRBP evolved by several gene duplications that occurred 600-800 million years ago, before the emergence of the vertebrates.     

Immunochemical mapping of antigenic regions on the human thyrotropin beta-subunit by antipeptide antibodies

To study antigenic sites present in the beta-subunit of human thyrotropin (hTSH), we produced site-specific antibodies directed against synthetic peptides analogous to the 1-18, 44-59, and 85-112 regions of the thyrotropin beta-subunit. The hTSH beta(1-18) peptide-carrier conjugate elicited antisera capable of binding to both radiolabeled hTSH and its beta-subunit whereas antibodies elicited against the hTSH beta(44-59) peptide-carrier conjugate bound only to the peptide. Thus, the NH2-terminal region of hTSH beta appears to be accessible at the surface of the hormone whereas the hTSH beta(44-59) region may be poorly accessible. Two monoclonal antipeptide antibodies that bound to 125I-hTSH beta, designated as TS01 and TS02, were selected after immunization with the hTSH beta(85-112) peptide-carrier conjugate. The antigenic site recognized by TS01 was located on the eight COOH-terminal(105-112) amino acid residues. TS02 antibody bound to an antigenic region included within Cys95 and Cys105. Both antigenic sites appeared to be more accessible on the free hTSH beta than on the hormone. Immunoblots performed on various preparations containing TSH revealed that TS02 antibody detected the beta-subunit from both the human and bovine species but not the rat TSH beta. Under reducing conditions, a low molecular weight material was identified in hTSH beta, likely caused by intrachain nicking.     

Isolation and sequencing of cDNAs and genomic DNAs encoding the alpha 4 chain of basement membrane collagen type IV and assignment of the gene to the distal long arm of human chromosome 2.

We cloned three overlapping cDNAs covering 2,452 base pairs encoding a new basement membrane collagen chain, alpha 4(IV), from rabbit corneal endothelial cell RNA. Nucleotide sequence analysis demonstrated that the clones encoded a triple-helical domain of 392 1/3 amino acid residues and a carboxyl non-triple-helical (NC1) domain of 231 residues. We also isolated a genomic DNA fragment for the human alpha 4(IV) chain, which contained two exons encoding from the carboxyl end of the triple-helical domain to the amino end of the NC1 domain. Identification of the clones was based on the amino acid sequence identity between the cDNA-deduced amino acid sequence and the reported amino acid sequence obtained from a fragment of the alpha 4(IV) collagen polypeptide M28+ (Butkowski, R. J., Shen, G.-Q., Wieslander, J., Michael, A. F., and Fish, A. J. (1990) J. Lab. Clin. Med. 115, 365-373). When compared with four other type IV collagen chains, the NC1 domain contained 12 cysteinyl residues in positions identical to those of the residues in those chains. The domain demonstrated 61, 70, 55, and 60% amino acid similarity with human alpha 1, human alpha 2, bovine alpha 3, and human alpha 5 chains, respectively. The human genomic DNA fragment allowed us to map the alpha 4(IV) gene (COL4A4) to the 2q35-2q37.1 region of the human genome.     

Sequence studies on the constant region of the Fd sections of rabbit immunoglobulin G of different allotype.

The amino acid sequence has been completed for the constant region of the Fd fragments of heavy chains from rabbit IgG (immunoglobulin G) of allotype Aa1 and Aa3. The amino acid sequence given by Fruchter et al. [(1970) Biochem. J. 116, 249-259] for the constant region of the Fd fragment from Aa1 IgG was extended and in in part corrected to give a continuous sequence of 140 residues. No allotype-related sequence variation was found in the constant section of the Fd fragment. This evidence confirms the view that the differences in sequence between the variable regions of Aa1 and Aa3 IgG [Mole et al., (1971) Biochem. J. 124, 301-318] are responsible for the allotypic specificities.     

Immune recognition of serum albumin.15. Localization by synthesis of antigenic site 4 of bovine serum albumin to the region around the disulfide bond 166-175

Recently, we have localized and confirmed by synthesis the regions within which reside five major antigenic sites of bovine serum albumin and proposed that the remaining sixth major antigenic site (antigenic site 4) was localized within subdomain 3 (fragment 115-184) of albumin. In the present work, antigenic site 4 was localized to fall around the disulfide bond 166-175 by synthesis and immunochemical reactivity of the region 162-179. The synthetic 18-residue peptide was shown to bind substantial amounts of antibodies from early (38-day) and late (398-day) anti-albumin antisera from two rabbits. As much as half the total anti-albumin antibodies could be bound by the peptide from the late antisera. It was concluded that antigenic site 4 resides within, but may not include all of, the region 162-179 of albumin.     

Identification of a neural cell specific variant of microtubule-associated protein 4

The microtubule-binding domain of MAP4, a ubiquitous microtubule-associated protein, contains a region rich in proline and basic residues (proline-rich region). We searched the bovine adrenal gland for MAP4 isoforms, and identified a novel variant lacking 72 consecutive amino acid residues within the proline-rich region, as compared with the full-length MAP4. The amino acid sequence of the missing region was highly conserved (about 85% identity/similarity) among the corresponding regions of bovine, human, mouse, and rat MAP4, which suggested the functional significance of this region. A comparison of the genomic sequence with the cDNA sequence revealed that the missing region is encoded by a single exon. A MAP4 variant cDNA homologous to the bovine form was also detected in rat cells, suggesting that the new variant can be generated by alternative splicing, not only in bovine but also in other mammalian species. The mRNA expression of the novel isoform was restricted to the brain and the adrenal medulla, suggesting that this isoform is specific to a certain cell type. Using a bacterially expressed fragment corresponding to the microtubule-binding domain of the novel isoform, we analyzed its in vitro characteristics. The fragment induced microtubule assembly and bound to preformed microtubules, but the activities were slightly lower than those of the conventional MAP4 fragment, which carries the full-length proline-rich region. The microtubules assembled in the presence of the fragment failed to be bundled. Instead, a constant spacing between neighboring microtubules was observed.     

Interaction of the 47-kDa talin fragment and the 32-kDa vinculin fragment with acidic phospholipids: a computer analysis.

In recent in vitro experiments, it has been demonstrated that the 47-kDa fragment of the talin molecule and the 32-kDa fragment of the vinculin molecule interact with acidic phospholipids. By using a computer analysis method, we determined the hydrophobic and amphipathic stretches of these fragments and, by applying a purpose-written matrix method, we ascertained the molecular amphipathic structure of alpha-helices. Calculations for the 47-kDa mouse talin fragment (residues 1-433; NH2-terminal region) suggest specific interactions of residues 21-39, 287-342, and 385-406 with acidic phospholipids and a general lipid-binding domain for mouse talin (primary amino acid sequence 385-401) and for Dictyostelium talin (primary amino acid sequence 348-364). Calculations for the 32-kDa chicken embryo vinculin fragment (residues 858-1066; COOH-terminal region) and from nematode vinculin alignment indicate for chicken embryo vinculin residues 935-978 and 1020-1040 interactions with acidic phospholipids. Experimental confirmation has been given for vinculin (residues 916-970), and future detailed experimental analyses are now needed to support the remaining computational data.     

The antibody response to myoglobin is independent of the immunized species. Analysis in terms of replacements in the antigenic sites and in environmental residues of the cross-reactions of fifteen myoglobins with sperm-whale myoglobin antisera raised in different species.

The recent determination of the entire antigenic structure of sperm-whale myoglobin with rabbit and goat antisera has permitted the examination of whether the antigenic structure recognized by antibodies depends on the species in which the antisera are raised. Also, by knowledge of the antigenic structure, the molecular factors that determine and influence antigenicity can be better understood in terms of the effects of amino acid substitutions occurring in the antigenic sites and in the environmental residues of the sites. In the present work, the myoglobins from finback whale, killer whale, horse, chimpanzee, sheep, goat, bovine, echidna, viscacha, rabbit, dog, cape fox, mouse and chicken were examined for their ability to cross-react with antisera to sperm-whale myoglobin. By immunoadsorbent titration studies with radioiodinated antibodies, each of these myoglobins was able to bind antibodies to sperm-whale myoglobin raised in goat, rabbit, chicken, cat, pig and outbred mouse. It was found that the extent of cross-reaction of a given myoglobin was not dependent on the species in which the antisera were raised. This indicated that the antibody response to sperm-whale myoglobin (i.e. its antigenic structure) is independent of the species in which the antisera are raised and is not directed to regions of sequence differences between the injected myoglobin and the myoglobin of the immunized host. Indeed, in each antiserum from a given species examined, that antiserum reacted with the myoglobin of that species. The extent of this auto-reactivity for a given myoglobin was comparable with the general extent of cross-reactivity shown by that myoglobin with antisera raised in other species. The cross-reactivities and auto-reactivities (both of which are of similar extents for a given myoglobin) can be reasonably rationalized in terms of the effects of amino acid substitutions within the antigenic sites and within the residues close to these sites. These findings confirm that the antigenicity of the sites is inherent in their three-dimensional locations.     

Identification and characterization of a phospholipid-binding site of bovine factor Va

Coagulation factor Va is a cofactor which combines with the serine protease factor Xa on a phospholipid surface to form the prothrombinase complex. The phospholipid-binding domain of bovine factor Va has been reported to be located on the light chain of the molecule and more precisely on a fragment of Mr = 30,000 which is obtained after digestion of factor Va light chain by factor Xa. This proteolytic fragment is located in the NH2-terminal part of factor Va light chain (residues 1564-1765). In order to further characterize the lipid-binding domain of bovine factor Va, isolated bovine light chain was preincubated with synthetic phospholipid vesicles (75% phosphatidylcholine, 25% phosphatidylserine) and digested with trypsin, chymotrypsin, and elastase. Two peptide regions protected from proteolytic cleavage were identified and characterized from each proteolytic digestion. A comparison of the NH2-terminal sequence and amino acid composition of the two tryptic peptides with the deduced sequence of human factor V indicates a match with residues 1657-1791 of the light chain of human factor V for one peptide and residues 1546-1656 for the other peptide. When chymotrypsin or elastase were used for digestion, the NH2-terminal sequence of one peptide showed a match with residues 1667-1797 of the light chain, while the other peptide presented an NH2-terminal sequence identical with the previously described for the bovine factor Va light chain. When these peptides were assayed for direct binding to phospholipid vesicles, only the tryptic and the chymotryptic peptides covering the middle region of the A3 domain of the bovine factor Va light chain demonstrated an ability to interact with phospholipid vesicles. Thus, knowing that the factor Xa cleavage site on the factor Va light chain is located between residues 1765 and 1766 of the light chain this lipid-binding region of the bovine factor Va is further localized to amino acid residues 1667-1765.     

Purification and properties of the manganese superoxide dismutase from the liver of bullfrog, Rana catesbeiana

A manganese superoxide dismutase (Mn-SOD) from the liver of bullfrog, Rana catesbeiana, was purified to electrophoretic homogeneity. The enzyme has a molecular weight of about 84,000 and is composed of four identical subunits, each containing one manganese atom. The amino acid composition of the enzyme is similar to that of Mn-SODs isolated from human and chicken livers, but differs considerably from that of the Escherichia coli enzyme (D. Barra et al. (1984) J. Biol. Chem. 259, 12595-12601; R. A. Weisiger and I. Fridovich (1973) J. Biol. Chem. 248, 3582-3592; H. M. Steinman (1978) J. Biol. Chem. 253, 8708-8720). The N-terminal amino acid is lysine. The sequence of 23 amino acid residues in the N-terminal region was determined. It shows excellent homologies with those of the human and chicken enzymes (H. M. Steinmam and R. L. Hill (1973) Proc. Natl. Acad. Sci. USA 70, 3725-3729; C. Ditlow et al. (1982) Carlsberg Res. Commun. 47, 81-91). The frog liver enzyme is also located exclusively in the mitochondrial matrix. Immunologically the same enzyme is also found in the tadpole liver, in an amount of about one-half of that in the adult bullfrog.