A radioimmunoassay for the Gm(b0) allotype of human immunoglobulins

A radioimmunoassay for the human allotype Gm(b⁰) which provides a sensitive and quantitative measurement of the level of this IgG3 genetic marker has been developed. The assay system can detect 15 nanograms of Gm(b⁰) IgG3 protein and is not inhibited by immunoglobulins of other allotypes and isotypes. Using this assay, good correlation was found between IgG3 and Gm(b⁰) levels in homozygous Gm(f, b⁰) sera and gene dosage effects could be confirmed. The correlation between Gm(b⁰) levels and IgG3 in Negroid Gm(a, b⁰) sera was not as good. This reduced correlation has been attributed to antigen differences in the IgG3 Gm markers characteristic of some Negroid Gm(a, b⁰) sera.     

Partial amino acid sequence in the N-terminal region of an anti-pneumococcal immunoglobulin heavy chain of allotype a2 (Short Communication)

The amino acid sequence of the N-terminal 48 residues of the heavy chain derived from a homogeneous rabbit antibody to type III pneumococci is described. This chain of allotype a(2) is compared with other rabbit heavy chains of allotypes a(1), a(2) and a(3). Within the N-terminal 25 positions, two chains which carry the same allotype a(2) possess identical amino acid sequences, but differ markedly from heavy chains of allotypes a(1) and a(3). Sequence variability is observed in residues 26-27 and 30-34, but not in residues 35-48.     

The frequencies of Gm(1), Gm(2), Gm(4), Gm(12), and Inv(1) in Hessen (Germany)

Summary The serum groups Gm(1) [Gm(a)], Gm(2) [Gm(x)], Gm(4) [Gm(f)]. Gm(12) [Gm(b)] and Inv(1) [Inv(1)] of 2000 sera of healthy blood donors from the land Hesse were examined. The results obtained were compared with those known until now. Three persons, not related to each other, possessed the extremely rare phenotype Gm(-1, 2, 4, 12) [Gm (a-x+b+f+)]. In 0.75% of the cases we found a discordant behaviour of the factors Gm(4) and Gm(12) [Gm(f) and Gm(b)].

---

Zusammenfassung 2000 Seren von gesunden Blutspendern aus Hessen wurden bezüglich der Gamma-Globulin-Serumgruppen Gm(1) [Gm(a)], Gm(2) [Gm(x)], Gm(4) [Gm(f)]. Gm(12) [Gm(b)] und Inv(1) [Inv(1)] untersucht. Die gefundenen Resultate wurden mit den bisher bekannten verglichen. Drei miteinander nicht verwandte Personen wiesen den äußerst seltenen Phänotyp Gm(-1, 2, 4, 12) [Gm(a-x+b+f+)] auf. In 0.75% der Fälle fanden wir ein diskordantes Verhalten der Faktoren Gm(4) und Gm(12) [Gm(f) und Gm(b)].

---

---

Director: Prof. Dr. W. Wachsmuth

---

Director: Prof. Dr. W. Spielmann

---

The nomenclature suggested by WHO at a round-table conference over genes, genotypes and allotypes of immunglobulins is used. The conference took place in Geneva on the 1965 31. 5. to the 5. 6. [5].

---

With technical assistance of S. Mohs.     

The nucleotide sequence of a human immunoglobulin C gamma1 gene

We report the nucleotide sequence of a gene encoding the constant region of a human immunoglobulin gamma 1 heavy chain (C gamma 1). A comparison of this sequence with those of the C gamma 2 and C gamma 4 genes reveals that these three human C gamma genes share considerable homology in both coding and noncoding regions. The nucleotide sequence differences indicate that these genes diverged from one another approximately 608 million years ago. An examination of hinge exons shows that these coding regions have evolved more rapidly than any other areas of the C gamma genes in terms of both base substitution and deletion/insertion events. Coding sequence diversity also is observed in areas of CH domains which border the hinge.     

The membrane-spanning segment of invariant chain (I gamma) contains a potentially cleavable signal sequence

The human invariant chain (I gamma) of class II histocompatibility antigens spans the membrane of the endoplasmic reticulum once. It exposes a small amino-terminal domain on the cytoplasmic side and a carboxy-terminal, glycosylated domain on the exoplasmic side of the membrane. When the exoplasmic domain of I gamma is replaced by the cytoplasmic protein chloramphenicol acetyltransferase (CAT), CAT becomes the exoplasmic, glycosylated domain of the resulting membrane protein I gamma CAT. Deletion of the hydrophilic cytoplasmic domain from I gamma CAT gives rise to a secreted protein from which an amino-terminal segment is cleaved, most likely by signal peptidase. We conclude that the membrane-spanning region of I gamma contains a signal sequence in its amino-terminal half and that hydrophilic residues at the amino-terminal end of a signal sequence can determine cleavage by signal peptidase.     

Structural studies of a human gamma 3 myeloma protein (Jir) bearing the allotypic marker Gm(st)

The authors established the amino acid substitutions determining G3m(s) and G3m(t) specificities, which characterize Mongoloid populations, by sequence analysis of the Fc region of a myeloma protein (Jir). By comparing the amino acid sequences of the IgG3 (Jir) and the other IgG subclasses analyzed to date, it was found that G3m(s) was an isoallotype specified by an amino acid substitution at position 435; i.e., whereas the subclasses IgG1, IgG2, and IgG4 had histidine in common, G3m(s-) had arginine in this position. This was also confirmed by the observation that the Fc fragment in question bound to protein A. It was also established that the amino acid at position 379 of G3m(t-) IgG3 and the other subclasses was valine, whereas methionine in this position was specific for G3m(t+). In addition, the amino acids at position 339 of G3m(u-) IgG3 Jir was threonine, and at position 296 of G3m(g-) IgG3 Jir was tyrosine. These findings are not in accord with the hitherto postulated relations of alanine and phenylalanine to G3m(u-) and G3m(g-), respectively. Finally, this study showed that a large number of substitutions occurred at positions 384 through 389, which suggests that many specificities of the G3m(b) group occur on IgG3 proteins.     

Partial amino acid sequence of a rabbit immunoglobulin light chain of allotype b5

The amino acid sequence of a rabbit immunoglobulin light chain of allotype b5 has been nearly completed. A comparison of its structure with that of light chains of allotypes b4, b6, and b9 confirms that the constant regions of these various kappa chains differ by 20-35%. The substitutions are clustered in parts of the second half of the chain, and the b5 form bears more resemblance to the b6 chain than to any other, in good agreement with previous serological data. The analysis of the variable region reveals the existence of certain allotype-associated residues which have also been reported in other b5 chains, but not in proteins of the other allotypes. An examination of the rabbit light chain sequences between positions 96 and 107 suggests that this portion of the chain may be encoded separately by a joining "J" DNA segment, as has been described previously for murine and human immunoglobulins. In the rabbit, however, these J kappa regions appear to differ from one allotype to another. Together with the extensive variations of the constant regions, these data suggest that the rabbit kappa gene organization more closely resembles the murine gamma system (four different C gamma genes each flanked by its J segment) than the murine kappa system (a single C kappa gene).     

Membrane-type matrix metalloproteinase-1 (MT1-MMP) is a processing enzyme for human laminin gamma 2 chain

Processing of the laminin-5 (Ln-5) gamma 2 chain by membrane-type-1 matrix metalloproteinases (MT1-MMP) promotes migration and invasion of epithelial and tumor cells. We previously demonstrated that MT1-MMP cleaves the rat gamma 2 chain at two sites, producing two major C-terminal fragments of 100 (gamma 2') and 80 (gamma 2 x) kDa and releasing a 30-kDa fragment containing epidermal growth factor (EGF)-like motifs (domain III (DIII) fragment). The DIII fragment bound the EGF receptor (EGF-R) and stimulated cell scattering and migration. However, it is not yet clear whether human Ln-5 is processed in a similar fashion to rat Ln-5 because one of the two MT1-MMP cleavage sites present in rat gamma 2 is not found in human gamma 2. To identify the exact cleavage site for MT1-MMP in human Ln-5, we purified both the whole molecule as well as a monomeric form of human gamma 2 that is frequently expressed by malignant tumor cells. Like rat Ln-5, both the monomer of gamma 2, as well as the gamma 2 derived from intact Ln-5, were cleaved by MT1-MMP in vitro, generating C-terminal gamma 2' (100 kDa) and gamma 2 x (85 kDa) fragments and releasing DIII fragments (25 and 27k Da). In addition to the conserved first cleavage site used to generate gamma 2', two adjacent cleavage sites (Gly(559)-Asp(560) and Gly(579)-Ser(580)) were found that could generate the gamma 2 x and DIII fragments. Two of the three EGF-like motifs present in the rat DIII fragment are present in the 27-kDa human fragment, and like the rat DIII, this fragment can promote breast carcinoma cell migration by engaging the EGF-R. These results suggest that MT1-MMP processing of Ln-5 in human tumors may stimulate the EGF-R, resulting in increased tumor cell scattering and migration that could possibly increase their metastatic potential.     

Characterization and DNA sequence of the b6w2 allotype of the rabbit immunoglobulin kappa 1 light chain (b locus)

The b6w2 allotype of the constant region of the rabbit immunoglobulin kappa 1 (k1) light chain (b locus) was discovered in wild populations from northern Spain. At the serological level, the b6w2 allotype is characterized by the presentation of all b6-specific epitopes, while an allotypic determinant which is shared between the nominal b5 and b6 allotypes is lacking. The DNA fragment encoding the b6w2 allotype was amplified by means of the polymerase chain reaction, and sequenced directly by dideoxy-DNA-sequencing. When compared with the sequence of the nominal b6 allele, the b6w2 sequence differs at eleven nucleotide positions (96.5% similarity). This variation corresponds to amino acid replacements at 1) the three positions C-terminal to the peptidyl junction with the variable region (amino acid positions 109–111);2) the four positions N-terminal to the interdomain disulfide bond (167–170); and 3) two positions in the vicinity of the interchain disulfide bond (190 and 210). The nature and distribution of the observed nucleotide substitutions strongly suggest a possible role of the extra interdomain disulfide bond in the unusual evolutionary dynamics of the rabbit K1 light chain.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number Z48308     

Amino acid sequence of the N-terminal aggregation and cross-linking region (7S domain) of the alpha 1 (IV) chain of human basement membrane collagen

The amino acid sequence of the 216-residue-long N-terminal aggregation and cross-linking 7S domain of the alpha 1 (IV) chain of human placental basement membrane collagen is presented. The N terminus of the alpha 1 (IV) chain starts with a non-triple-helical region, which is at least 15 residues long and contains four cysteine and two lysine residues as putative cross-linking sites. This segment is followed by a 120-residue-long triple helical region, which contains the unusual occurrence of a cysteine residue in the Xaa position of a Gly-Xaa-Yaa triplet. Since individual molecules in the 7S domain are associated in an antiparallel manner, this cysteine probably aligns with one of the four cysteines in the amino-terminal end of an adjacent molecule, forming an intermolecular disulfide bridge. The length of the overlap of two adjacent molecules is estimated to be about 110 residues. The triple helix adjacent to the overlap zone is interrupted by a 10-residue-long non-helical area, which is probably responsible for the flexible region of the molecules in the neighbourhood of the overlap zone observed in the electron microscope. The mode of aggregation of the 7S domain, the formation of intermolecular cross-links as well as the relatively high stability of this region against proteolytic attack are discussed in the light of the elucidated amino acid sequence.