Gene conversion in human immunoglobulin gamma locus shown by unusual location of IgG allotypes

The constant region of the gamma 1, gamma 2 and gamma 3 heavy chains of the human IgG1, IgG2 and IgG3 immunoglobulins carries antigenic determinants or G1m, G2m and G3m allotypes, which are genetic markers of these subclasses. The exceptional presence on gamma 1 and gamma 2 chains of Gm allotypes usually located on the CH3 domain of gamma 3 shows an unexpected clustering of base changes and subsequent identity of short DNA sequences in the CH3 exon of the non-allelic gamma 1, gamma 2 and gamma 3 genes. Such clusters of substitutions are not easily explained on the classical basis of point mutations. A gene conversion, which substituted a segment of the gamma 1 or gamma 2 gene with the homologous region of the non-allelic gamma 3 gene, is more likely. Other examples of possible conversion involving the gamma genes are described. The conservation or the restoration of short sequences produced by the conversion events might be related to the biological properties of the constant region of the heavy chains.     

Baboon immunoglobulin constant region heavy chains: identification of four <Emphasis Type="Italic">IGHG</Emphasis> genes

The increasing use of nonhuman primate models in biomedical research and especially in vaccine development requires the characterization of their immunoglobulin genes and corresponding products. Therefore, we sequenced, cloned and characterized the four immunoglobulin gamma chain constant region genes ( IGHG) present in baboons. These four genes were designated IGHG1, IGHG2, IGHG3 and IGHG4 on the basis of sequence similarities with the four human genes encoding the IgG1, IgG2, IgG3 and IgG4 subclasses and the three known rhesus macaque IGHG genes. Specifically, the baboon IgG1, IgG2, IgG3 and IgG4 sequences exhibit 90.3%, 88.3%, 86.7% and 89.6% amino acid identity to their human counterpart. The percent of amino acid identity of baboon IgG1, IgG2 and IgG3 to the corresponding rhesus macaque sequences is 98.5, 93.1 and 94.4, respectively. Therefore, baboon and rhesus macaque IGHG genes are highly homologous to each other. The majority of differences existing between baboon and human sequences are clustered in the hinge region, with the upper hinge being the most diverse and containing several proline residues. Similar to rhesus macaques, the hinge regions of all baboon IGHG genes consist of a single exon, whereas in humans the IgG3 molecule is encoded by multiple exons. These results confirm the evolutionary instability of the hinge region and indicate that functional properties associated with the hinge regions of baboon and human IgG molecules might differ between the two species.     

Chicken immunoglobulin gamma-heavy chains: limited VH gene repertoire, combinatorial diversification by D gene segments and evolution of the heavy chain locus

cDNA clones encoding the variable and constant regions of chicken immunoglobulin (Ig) gamma-chains were obtained from spleen cDNA libraries. Southern blots of kidney DNA show that the variable region sequences of eight cDNA clones reveal the same set of bands corresponding to approximately 30 cross-hybridizing VH genes of one subgroup. Since the VH clones were randomly selected, it is likely that the bulk of chicken H-chains are encoded by a single VH subgroup. Nucleotide sequence determinations of two cDNA clones reveal VH, D, JH and the constant region. The VH segments are closely related to each other (83% homology) as expected for VH or the same subgroup. The JHs are 15 residues long and differ by one amino acid. The Ds differ markedly in sequence (20% homology) and size (10 and 20 residues). These findings strongly indicate multiple (at least two) D genes which by a combinatorial joining mechanism diversify the H-chains, a mechanism which is not operative in the chicken L-chain locus. The most notable among the chicken Igs is the so-called 7S IgG because its H-chain differs in many important aspects from any mammalian IgG. The sequence of the C gamma cDNA reported here resolves this issue. The chicken C gamma is 426 residues long with four CH domains (unlike mammalian C gamma which has three CH domains) and it shows 25% homology to the chicken C mu. The chicken C gamma is most related to the mammalian C epsilon in length, the presence of four CH domains and the distribution of cysteines in the CH1 and CH2 domains. We propose that the unique chicken C gamma is the ancestor of the mammalian C epsilon and C gamma subclasses, and discuss the evolution of the H-chain locus from that of chicken with presumably three genes (mu, gamma, alpha) to the mammalian loci with 8-10 H-chain genes.     

Studies on the Fc gamma receptor of the murine macrophage-like cell line P388D1. II. Binding of human IgG subclass proteins and their proteolytic fragments.

Binding studies of human IgG proteins to murine P388D1 cells indicated that they bind to an apparently homogeneous Fc receptor population. The association constant was 0.89 x 10(6)M-1 at 22 degrees C and was comparable to the binding affinities of homologous murine IgG2a and IgG2b. The number of receptor sites was found to be approximately 6 x 10(5)/cell. Fc gamma 1 and Fc gamma 3 fragments bound with an affinity comparable to that of the parent proteins. The P388D1 receptors could discriminate between the human IgG subclasses; the relative cytophilic activity was IgG3 greater than IgG1 greater than IgG4 and IgG2 was devoid of binding activity. Fragments corresponding to the C gamma 2 and C gamma 3 domains of human IgG1 were both unable to bind to the P388D1 receptors either alone or in equimolar combination. This suggests that the cytophilic site may be formed cooperatively by interaction between the two domains. The integrity of the hinge region appeared to be essential for full expression of cytophilic activity since reduction of the hinge-region disulfides in both human IgG1 and its Fc fragment markedly decreased their binding affinity. In addition, a mutant IgG1 molecule lacking the hinge region was significantly less cytophilic than its normal counterpart.     

The complete amino acid sequence of monkey progastricsin

The complete amino acid sequence of progastricsin from the Japanese monkey (Macaca fuscata) was determined. Progastricsin is composed of 374 residues, including the gastricsin moiety of 331 residues and the activation segment of 43 residues. Upon activation under acidic conditions, progastricsin was converted to gastricsin via the intermediate protein species. NH2-terminal sequence determination of these protein species enabled us to deduce the NH2-terminal 78-residue sequence of progastricsin, including the 43-residue activation segment. The complete sequence of the gastricsin moiety was determined using peptide fragments obtained by several chemical and enzymatic cleavages. The molecular weight of progastricsin was determined to be 40,785. As compared with pepsinogen A of the same monkey species, deletion of 4 residues and insertion of 5 residues were observed. Although monkey progastricsin and pepsinogen A have highly homologous sequences around the two active site aspartyl residues, the homology between these proteins is rather small (49% identity). This indicates that progastricsin diverged from pepsinogen A in the early phase of the evolution of gastric aspartyl proteinases.     

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.     

The crystallizable human myeloma protein Dob has a hinge-region deletion.

During experiments to prepare heavy-metal derivatives of the crystallizable human IgG1 (k) immunoglobulin Dob, it became apparent that this protein has several unusual features. (1) Instead of the four labile interchain disulfide bridges ordinarily found in IgG1, the Dob protein has only a single interchain disulfide bridge, which connects its two light chains. (2) The Dob heavy chain appears to be slightly smaller than a control gamma1 chain, as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and by gel filtration in guanidine. (3) The Dob heavy chain has three fewer residues of half-cystine than expected in gamma1 chains. (4) The Dob IgG is relatively resistant to digestion with papain and trypsin; however, it is readily digested with pepsin, although at an unusual site. These findings suggest that some or all of the gamma1 hinge region is missing in Dob. To localize the deletion, we prepared an F(ab')2 fragment consisting of two heavy-chain pieces (Fd') noncovalently associated with the light-chain dimer. The Fd' piece was isolated and digested with trypsin. The sequence of the C-terminal tryptic peptide was Val-Ala-Pro-Glu-Leu-Leu-Gly-Gly-Pro-Ser-Val. Positions 2-11 of this peptide are identical with residue positions 231-240 of the gamma1 chain. The N-terminal valine could be either Val-211 or Val-215 of the gamma1 sequence. A tryptic peptide, Val-Asp-Lys-Lys, was also isolated from Dob Fd'; this sequence is not found in the variable region of the Dob heavy chain [Steiner, L. A., Garcia Pardo, A., & Margolies, M. N. (1979) Biochemistry (following paper in this issue)] but corresponds to positions 211-214 of the gamma1 constant region. Therefore, the deletion cannot include these residues and must begin after Val-215; normal gamma1 sequence resumes at Ala-231. The same 15-residue deletion has been found in two other IgG1 proteins, Mcg [Fett, J. W., Deutsch, H. F., & Smithies, O. (1973) Immunochemistry 10, 115] and Lec [Rivat, C., Schiff, C., Rivat, L., Ropartz, C., & Fougereau, M. (1976) Eur. J. Immunol. 6, 545]. Possible explanations for the occurrence of identical hinge-region deletions in three different immunoglobulins are suggested by recent experiments demonstrating that the three constant domains and the hinge region of mouse gamma1 chains are each encoded by separate segments of DNA [Sakano, H., Rogers, J. H., Hüppi, K., Brack, C., Traunecker, A., Maki, R., Wall, R., & Tonegawa, S. (1979) Nature (London) 277, 627].     

The amino acid sequence of a human immunoglobulin G3m(g) pFc' fragment.

Amino acid sequence analysis of the pFc' fragment obtained by pepsin digestion of an IgG3; G3m(g) human myeloma protein HER shows it to consist of 112 residues. It starts at position 334 (gamma1 numbering), contains eight residues of the Cgamma2 region, and the whole Cgamma3 domain. Comparison with the sequence of gamma1 shows five differences including an extra Met at 397. Each is accountable by a single base substitution. The sequence is identical to that of a G3m(b0) molecule except for the previously noted allotype related Tyr/Phe exchange at position 436. The high degree of homology (95%) among gamma-chain subclasses suggests a recent diversification.     

Staphylococcus aureus isolated from Kawasaki disease patients hyper-releases extracellular protein A.

S. aureus isolates from patients with Kawasaki disease (KD) release high levels of extracellular protein A (SpA), as compared to S. aureus in other diseases. The molecular weight of this released protein A is about 70 kDa. Extracellular KD SpA purified by affinity chromatography possessed the same amino acid sequence at the NH2-terminal IgG binding region and the same antigenic specificity as recombinant and cell-wall-bound SpA preparations. The size of DNA fragments containing the spa gene from S. aureus KD strains was 160-165 kb. All of these DNA fragments contained the igb portion encoding the IgG-binding region of KD SpA. Significantly higher molecular size of the SpA molecules hyper-released in the stationary-phase culture and the lack of production of other exo-proteins allow us to speculate that S. aureus isolated from patients with KD have mutations occurring in the agr locus.     

Amino acid sequence around a "hinge" region and its "autophosphorylation" site in bovine Lung cGMP-dependent protein kinase

An exposed "hinge" region of cGMP-dependent protein kinase is known to be susceptible to both limited proteolysis and autophosphorylation. A 91-residue fragment has been isolated from this region and its amino acid sequence has been compared with the analogous regions of the cAMP-dependent protein kinases. Although a resemblance among these sequences is not striking, the phosphorylation sites are in corresponding regions toward the NH2 termini, and there are indications of homology in the vicinity of their autophosphorylation sites. As in the cAMP-dependent protein kinase, the site of autophosphorylation and the site of susceptibility to limited proteolysis are very near each other in the primary structure. The actual site of autophosphorylation (the underlined threonine residue in Pro-Arg-Thr-Thr-Arg) is quite different from those in the regulatory subunit of Type II cAMP-dependent kinase or the site in Type I regulatory subunit that can be phosphorylated by the cGMP-dependent protein kinase.     

Isolation and sequencing of a cDNA clone encoding acid phosphatase in rat liver lysosomes

A full length cDNA for acid phosphatase in rat liver lysosomes was isolated and sequenced. The predicted amino acid sequence comprises 423 residues (48,332 Da). A putative signal peptide of 30 residues is followed by the NH2-terminal sequence of lysosomal acid phosphatase (45,096 Da). The deduced NH2-terminal 18-residue sequence is identical with that determined directly for acid phosphatases purified from the rat liver lysosomal membranes. The primary structure deduced for acid phosphatase contains 9 potential N-glycosylation sites and a hydrophobic region which could function as a transmembrane domain. It exhibits 89% and 67% sequence similarities in amino acids and nucleic acids, respectively, to human lysosomal acid phosphatase. The amino acid sequence of the putative transmembrane segment shows a complete similarity to that of the human enzyme. Northern blot hybridization analysis identified a single species of acid phosphatase mRNA (2.2 kbp in length) in rat liver.     

Molecular cloning and nucleotide sequence of the cDNA for human liver glutamate dehydrogenase precursor

Two cDNA clones (lambda GDHh1 and lambda GDHn61) for glutamate dehydrogenase (GDH) were isolated from a human liver cDNA library in lambda gt11. The clone, lambda GDHh1, was isolated from the library using a synthetic 45mer oligodeoxy-ribonucleotide, the sequence of which was derived from the known amino acid sequence near the NH2-terminus of human liver GDH. Subsequently, lambda GDHn61 was isolated from the same library using lambda GDHh1 as a probe. The inserts of both clones contained an overlapping cDNA sequence for human liver GDH, consisting of a 5'-untranslated region of 70 bp, an open reading frame of 1677 bp, a 3'-untranslated region of 1262 bp and a 15 base poly(A) tract. The predicted amino acid sequence revealed that the human liver GDH precursor consisted of a total of 558 amino acid residues including the NH2-terminal presequence of 53 amino acids. The sequence deduced for the mature enzyme showed 94% homology to the previously reported amino acid sequence of human liver GDH.     

The high affinity calcium-binding site involved in protein C activation is outside the first epidermal growth factor homology domain.

Binding Ca2+ to a high affinity site in protein C and 4-carboxyglutamic acid (Gla)-domainless protein C results in a conformational change that is required for activation by the thrombin-thrombomodulin complex, the natural activator of protein C. It has been hypothesized that this high affinity Ca(2+)-binding site is located in the NH2-terminal epidermal growth factor (EGF) homology region of protein C. We have expressed in human 293 cells a deletion mutant of protein C (E2-PD) which lacks the entire Gla region as well as the NH2-terminal EGF homology region of protein C. Ca2+ inhibits activation of E2-PD or Gla-domainless protein C by thrombin with half-maximal inhibition occurring at Ca2+ concentrations of 103 +/- 11 and 70 +/- 7 microM, respectively, but is required for both E2-PD and Gla-domainless protein C activation by the thrombin-thrombomodulin complex with half-maximal acceleration occurring at Ca2+ concentrations of 87 +/- 8 and 89 +/- 8 microM, respectively. Both E2-PD and Gla-domainless protein C exhibit a reversible, Ca(2+)- but not Mg(2+)-dependent decrease (6 +/- 1%) in fluorescence emission intensity with Kd = 38 +/- 3 microM Ca2+. We conclude that the high affinity Ca(2+)-binding site important for the activation of protein C is located outside of the NH2-terminal EGF homology region and that the metal-binding site in the NH2-terminal EGF homology region may not be a high affinity site in intact protein C.     

Fasciola hepatica: parasite-secreted proteinases degrade all human IgG subclasses: determination of the specific cleavage sites and identification of the immunoglobulin fragments produced

The study was focused on the relationship of Fasciola hepatica-secreted proteinases and human IgG subclasses. Each IgG was incubated at different pH values and lengths of time with either the adult parasite excretion-secretion products or the purified cysteinyl proteinases cathepsin L1 and cathepsin L2. The Ig fragments produced were isolated and characterized by Western blot analysis, and the specific cleavage sites were determined by amino acid sequence analysis. Parasite excretion-secretion products and both cathepsins L produced similar degradation patterns and cleaved all human IgG subclasses at the hinge region, yielding at pH 7.3 and 37 degrees C Fab and Fc fragments in the case of IgG1 and IgG3 or Fab(2) and Fc in IgG2 and IgG4. While IgG1 and IgG3 were readily degraded by E/S products either in the presence or in the absence of reducing agents, IgG2 and IgG4 were resistant to proteolysis and were only digested in the presence of 0.1 M dithiothreitol. The cathepsins L needed the presence of dithiothreitol to digest IgG1, IgG2, and IgG4 whereas IgG3 was identically cleaved under both reducing and nonreducing conditions. The main cleavage sites produced by E/S products, CL1, or CL2 were located at the positions peptide bonds: His237-Thr238, Glu237-Cys239, Gly233-Asp234, and Ser241-Cys242 for gamma1, gamma2, gamma3, or gamma4, respectively. The enzymes gave additional splitting sites on the middle hinge of IgG3 to produce shorter Fc fragments and also produce Fd degradation of the IgG4. No cleavage specificity differences were found between CL1 and CL2, but they differed in the kinetics of IgG3 degradation. By lowering the pH, only the E/S products produced concomitant destruction of the Fc while preserving the Fab portion. Under all the conditions assayed the enzymes produced an Fc'-like fragment of 14-15 kDa corresponding to the whole CH3 domain of the immunoglobulin. Contrary to the extensive degradation produced by cathepsins on digested proteins, its actions on IgG subclasses were specific and restricted; thus, all the fragments produced could be potentially involved in the mechanisms used by the parasite to evade the host immune response.     

Primary structure of rat pulmonary surfactant protein D. cDNA and deduced amino acid sequence.

Surfactant protein D (SP-D) is a carbohydrate-binding glycoprotein containing a collagen-like domain that is synthesized by alveolar type II epithelial cells. The complete primary structure of rat SP-D has been determined by sequencing of a cloned cDNA. The protein consists of three regions: an NH2-terminal segment of 25 amino acids, a collagen-like domain consisting of 59 Gly-X-Y repeats, and a COOH-terminal carbohydrate recognition domain of 153 amino acids. There are 6 cysteine residues present in rat SP-D: 2 in the NH2-terminal noncollagenous segment and 4 in the COOH-terminal carbohydrate-binding domain. The collagenous domain contains one possible N-glycosylation site. The protein is preceded by a cleaved, NH2-terminal signal peptide. SP-D shares considerable homology with the C-type mammalian lectins. Hybridization analysis demonstrates that rat SP-D is encoded by a 1.3-kilobase mRNA which is abundant in lung and highly enriched in alveolar type II cells. Extensive homology exists between rat SP-D and bovine conglutinin.     

Purification and amino-terminal sequence of the bovine cardiac sodium-calcium exchanger: evidence for the presence of a signal sequence

The Na(+)-Ca2+ exchange carrier was purified from bovine cardiac tissue by a new procedure which relies principally upon anion-exchange chromatography. The purified protein exhibited two major bands on sodium dodecyl sulfate gels, at 120 and 160 kDa. The relative intensities of the two bands could be altered by variations in the procedures used for preparing the samples for electrophoresis, suggesting that they represent two different conformational states of the same protein. The NH2-terminal amino acid sequences of the 120- and 160-kDa bands were identical and agreed closely with a region of the deduced amino acid sequence of the recently cloned canine cardiac exchanger. The NH2-terminal sequence was preceded in the deduced sequence by a 32-residue segment that exhibited the characteristics of a signal sequence; the initial amino acid in the NH2-terminal sequence followed immediately after the predicted cleavage site for the signal sequence. The Na(+)-Ca2+ exchanger appears to be unique among membrane transport carriers in encoding a cleaved signal sequence. The characteristics of the sequences flanking the first putative transmembrane segment of the mature exchanger suggest that the signal sequence is necessary to ensure the correct topological orientation of the exchanger in the membrane.     

Amino-terminal sequence of chicken preproalbumin

Poly(A)-containing RNA was isolated from chicken liver and translated in a reticulocyte lysate protein-synthesizing system in the presence of radiolabeled amino acids. Chicken albumin was isolated from the translation products by immunoprecipitation and subjected to automated Edman radiosequencing. Comparison with the sequence of proalbumin showed that the translocation product (preproalbumin) contains an NH2-terminal extension of 18 amino acid residues. The NH2-terminal sequence of chicken preproalbumin was as follows: Met-18-Lys-Asn-Val-15-Thr-Leu-Ile-Ser-Phe-10-Ile-Phe-Leu-Phe-Ser-5-Ser-Ala-Thr- Ser-1-Arg1, where Arg1 represents the NH2-terminal residue of proalbumin. This NH2-terminal extension is very rich in hydrophobic amino acid residues and is similar to the signal sequences found in other secreted proteins. The signal sequence of chicken preproalbumin shows considerable homology with the signal sequences of rat and bovine preproalbumins, but little homology with the signal sequences of other chicken preproteins.     

Studies of the domain structure of mammalian DNA polymerase beta. Identification of a discrete template binding domain

Characterization of the domain structure of DNA polymerase beta is reported. Large scale overproduction of the rat protein in Escherichia coli was achieved, and the purified recombinant protein was verified by sequencing tryptic peptides. This protein is both a single-stranded DNA binding protein and a DNA polymerase consisting of one polypeptide chain of 334 amino acids. As revealed by controlled proteolysis experiments, the protein is organized in two relatively protease-resistant segments linked by a short protease-sensitive region. One of these protease-resistant segments represents the NH2-terminal 20% of the protein. This NH2-terminal domain (of about 75 residues) has strong affinity for single-stranded nucleic acids. The other protease-resistant segment, representing the COOH-terminal domain of approximately 250 residues, does not bind to nucleic acids. Neither domain, tested as purified proteins, has substantial DNA polymerase activity. The results suggest that the NH2-terminal domain is principally responsible for the template binding activity of the intact protein.