Amino acid sequence of the variable region of the light (lambda) chain from human myeloma cryoimmunoglobulin IgG Hil.

We have determined the complete amino acid sequence of the variable region of the light (lambda) chain from a human myeloma cryoimmunoglobulin (IgG Hil), the Fab fragment from which has been previously crystallized. The presence of unblocked alpha-amino terminal residue and the isolation of a CNBr fragment starting at position 46 and of a maleylated tryptic fragment spanning residues 61 to 189 provided three suitable starting points for automatic Edman degradation. In addition, tryptic peptides and chymotryptic subpeptides covering the whole extension of the light chain were obtained and characterized to further verify the sequence of the variable region and the established sequence of the constant region. The proposed sequence of the variable region indicates that it may be assigned to subgroup III. Positions 152 (serine) and 189 (arginine) correspond to the isotypic markers Kern- and Oz-, respectively. In addition, a novel substitution has been detected in the constant region where at position 155 isoleucine replaces the usually occurring valine.     

Amino acid sequence of galactosamine-containing glycopeptides in the hinge region of a human immunoglobulin D

Amino acid sequence and the location of seven galactosamine oligosaccharide moieties of the hinge region of the δ chain of human IgD NIG-65 have been determined. These oligosaccharide moieties are distributed in two distinct fashions: 1) three clusters each consisting of five amino acid residues with two consecutive attachment sites either Ala-X-Ala-Ser-Ser or Ala-X-Ala-Thr-Thr, where X can be any amino acid including proline, 2) one triplet sequence Val-Pro-Thr with one attachment site. We propose two rules with regard to the acceptor sequence for galactosamine oligosaccharides, the quintet sequence rule and triplet sequence rule.     

Amino acid sequence of the heavy-chain variable region of the crystallizable human myeloma protein Dob.

The amino acid sequence of the heavy-chain variable region of the crystallizable human myeloma protein Dob has been determined. This protein has previously been shown to have a deletion in the hinge region [Lopes, A. D., & Steiner, L. A. (1973) Fed. Proc., Fed. Am. Soc. Exp. Biol. 32, 1003; Steiner, L. A., & Lopes, A. D. (1979) Biochemistry (preceding paper in this issue)]. The complete sequence was established by analysis, in the automated sequenator, of the intact Fd' piece and of three large overlapping fragments prepared from Fd' by digestion with cyanogen bromide, by tryptic digestion of the citraconylated Fd', and by cleavage with hydroxylamine. Portions of the sequence were confirmed by examination of the amino acid composition and the partial sequence of a variety of small peptides obtained by enzymatic degradation. The Dob heavy-chain variable region appears to belong to the VHIII subgroup, but there are several unusual substitutions. Residue 45 in the Dob sequence is proline, although all other known heavy-chain sequences in man, mouse, rabbit, and guinea pig have leucine at this position. Positions 10 (aspartic acid), 68 (alanine), and 82 (leucine) in the Dob sequence are also atypical. There is no deleted segment in the variable region of the Dob heavy chain nor any abnormality in the variable-constant joining region. The hinge-region deletion appears to be the only gross structural anomaly in the Dob heavy chain.     

Amino acid sequence and location of the three glycopeptides in the Fc region of human immunoglobulin D

Amino acid sequence and the location of three glycopeptides of the Fc fragment obtained by limited proteolysis with trypsin of an intact myeloma protein(NIG-65) has been determined. These glycopeptides have glucosamine oligosaccharides(designated GlcN) which are attached to Asn-68, Asn-159, and Asn-210 by N-glycosidic linkages. Of these, Glcn-159 is characteristic of the δ chain and has no counterpart position in any of the other classes. On the other hand, GlcN-68 is shared by γ, μ, and ? chains, and GlcN-210 by α and μ chains, respectively. Although different classes of human immunoglobulins differ in the number and the kinds of oligosaccharides, the sites are often homologous and are related to the basic immunoglobulin domain structure.     

Primary structure of a human IgA1 immunoglobulin. V. Amino acid sequence of a human IgA lambda light chain (Bur).

The sequence of the lambda light chain of the Bur IgA1 molecule has been determined. It comprises 214 amino acid residues with a blocked NH2 terminus and lacks carbohydrate. The V-region sequence is of the VlambdaII subgroup and contains the coupled interchanges Arg-7 and Cys-87. The Lv3 region is comparatively short and hydrophobic in nature and lends support for the designation of this area as a hypervariable deletion region. The C-region exhibits the Mcg+ Kren+ Oz- isotypes. These appear coupled with substitution at position 100 (in the V-region). The pattern of nonrandom association of V- and C-regions and H and L chains is discussed in terms of the generation of antibody diversity. With the companion papers in this series, the complete primary structure of a human IgA1 molecule is established.     

Human immunoglobulin variable region genes: A new VH sequence used to detect polymorphism

A human heavy chain variable region subgroup III pseudogene (HV3.3) was isolated, characterized, and sequenced. When HV3. was hybridized to Southern blots of human DNA, two potentially informative polymorphic bands, resulting from 2.7 kb Hind III (HH2.7) and 7.3 kb Eco RI(HE7.3) fragments, were detected with frequencies of 0.553 and 0.606, respectively. These polymorphic bands showed Mendelian segregation in families and appeared to be in tight linkage disequilibrium with each other ( ₁ ² =24.91, P<0.001). Evidence from sibling-pair data indicated linkage of the Hind III polymorphic band to constant region allotypic and restriction fragment length polymorphism markers. Bands representing alternative forms of the polymorphic restriction sites were not detected for either HH2.7 or HE7.3. This indicates either that the alternative fragments comigrate with homologous fragments resulting from conserved restriction sites, or that the polymorphism is due to a gene duplication or deletion. No band segregating with HH2.7 was found in separate digests using eight other enzymes. Although this indicates that a major deletion is unlikely, it does not exclude the possibility of a gene deletion or duplication affecting the intergenic region(s) of one or more homologous genes. Whatever the precise explanation, these findings support the hypothesis that there is polymorphic variation of V _H gene repertoires in man.Previous address     

Amino acid sequence of normal (microheterogeneous) porcine immunoglobulin lambda chains.

The partial amino acid sequence of pooled, microheterogeneous pig immunoglobulin lambda chains was determined previously (Fran?k, F. (1970), FEBS Lett. 8, 269; Novotny, J., and Fran?k, F. (1975), FEBS Lett. 58, 24). In the present study, citraconylated pig lambda chains were digested by trypsin under conditions in which some of the epsilon-amino groups of lysine residues unmask. The resulting fragments were purified by gel filtration and ion-exchange chromatography at pH 3.0 in buffers containing urea; some of the fragments were found to be of intermediate size (i.e., larger than normal tryptic peptides but smaller than "citraconyl" peptides), thus permitting overlap information and amino acid sequences of all the 14 tryptic peptides to be deduced from amino acid compositions and partial amino acid sequences of selected fragments. In addition to completing the major amino acid sequence of pig immunoglobulin lambda chains, the present study demonstrates that it is possible to sequence microheterogeneous proteins with a suitable fragmentation strategy.     

Amino acid sequence of the human fibronectin receptor

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+-binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors.     

Amino acid sequence of the first 65 residues of IgA myeloma protein

Amino acid sequence of the first 65 residues of human IgA myeloma protein was determined. MH₂-terminal fragment was obtained by specific scission at Asn-Gly bond with NH₂OH from completely reduced and alkylated protein. Peptides were obtained by tryptic and chymotryptic digestions of the fragment following column chromatography in a volatile buffer system. After purification by high voltage electrophoresis, peptides were sequenced by manual Edman-dansyl technique. The fragment consists of 65 amino acid residues and has the sequence characteristic of V_HIII subgroup of heavy chains.     

Amino acid sequence of the a subunit of human factor XIII

Factor XIII is a plasma protein that plays an important role in the final stages of blood coagulation and fibrinolysis. The complete amino acid sequence of the a subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gtll cDNA library prepared from human placenta mRNA was screened with an affinity-purified antibody against the a subunit of human factor XIII and then with a synthetic oligonucleotide probe that coded for a portion of the amino acid sequence present in the activation peptide of the a subunit. Six positive clones were identified and shown to code for the a subunit of factor XIII by DNA sequence analysis. A total of 3831 base pairs was determined by sequencing six overlapping cDNA clones. This DNA sequence contains a 5' noncoding region or a region coding for a portion of a pro-piece or leader sequence, the mature protein (731 amino acids), a stop codon (TGA), a 3' noncoding region (1535 nucleotides), and a poly(A) tail (10 nucleotides). When the a subunit of human factor XIII was digested with cyanogen bromide, 11 peptides were isolated by gel filtration and reverse-phase HPLC. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 363 amino acid residues were identified. These amino acid sequences were in complete agreement with those predicted from the cDNA. The a subunit of factor XIII contained the active site sequence of Tyr-Gly-Gln-Cys-Trp, which is identical with that of tissue transglutaminase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of a cystatin from venom of the African puff adder (Bitis arietans).

The amino acid sequence of a cystatin from the venom of the African puff adder (Bitis arietans) is reported. It shows the protein to be more closely related to the Type 2 cystatins than the others, and yet it is only 31-37% identical to the known Type 2 cystatins, and differs strikingly in the insertion of a six-residue segment.     

Amino acid sequence of Escherichia coli biotin carboxyl carrier protein (9100).

The amino acid sequence of a proteolytic fragment of Escherichia coli biotin carboxyl carrier protein was determined from the structures of overlapping tryptic, thermolytic, and staphylococcal protease peptides together with automated sequenator analyses on the intact protein. The fragment, 82 residues in length, contains the single residue of biocytin of the protein. The relationship of the Mr = 9100 fragment to the native Mr = 22,500 subunit is discussed.     

The amino acid sequences of the three heavy chain constant region domains of a human IgG2 myeloma protein.

The amino acid sequences of most of the CH1, CH2 and CH3 domains of IgG Zie, a myeloma protein belonging to the IgG2 subclass, are presented. These data make possible a comparison of the sequences of residues 253-446 of all four subclasses of immunoglobulins: these residues make up almost the entire Fc regions. A comparison can also be made of the CH1 domain of IgG1 Eu and the CH1 domain of IgG2 Zie. Earlier sequence analyses of the Fc regions of subclass 1 and 3 proteins, and parts of the Fc regions of subclass 2 and 4 proteins showed that about 95% of these sequences were identical. The extended comparisons made possible by the data presented here show that this very high degree of identity is maintained throughout the four subclasses. Similarly, the CH1 domains of gamma 1 and gamma 2 chains were found to have about 93% sequence identity. It is unlikely that the few single amino acid changes within the constant region domains can account for the marked differences between subclasses observed in the region domains can account for the marked differences between subclasses observed in the biological effector functions of immunoglobulin Fc regions, especially since most of the changes are highly conservative. Rather, it seems probable that these functional differences are caused by conformational differences between the subgroups, which result from sequence differences in the hinge regions.