Adaptive evolution of the IgA hinge region in primates

IgA is a major component that prevents the penetration of pathogenic bacteria into mucosal surfaces. The IgA antibody is cleaved at the IgA hinge region with high specificity by IgA-specific proteases produced by several pathogenic bacteria. We conducted a genomic sequence analysis of the IgA genes of a wide spectrum of primates, including the first intron and second exon, which consist of the hinge region and the CH2 domain, to find evidence of positive selection. Because the hinge region is quite small, we combined the largest collection of sequences that could be clearly aligned and evaluated the total numbers of synonymous and nonsynonymous substitutions on the phylogenetic tree. The nonsynonymous to synonymous substitution ratio (d(N)/d(S) test) showed that hominoids, Old World monkeys, and New World monkeys have d(N)/d(S) ratios of 5.4, 6.3, and 4.2, respectively. Fisher's exact probability tests showed statistical significance for the Old World monkey. Because the substitution rates of the flanking sequences are more or less similar to the synonymous rates of the hinge region, these high values of d(N)/d(S) should be the result of positive selection at the hinge region. Combining the high sequence variability in each population and the highly accelerated nonsynonymous substitution rates in the hinge region, we conclude that this unusual IgA evolution is a molecular evidence of adaptive evolution possibly caused by the host-parasite relationship.     

Determination of aberrant O-glycosylation in the IgA1 hinge region by electron capture dissociation fourier transform-ion cyclotron resonance mass spectrometry

In a number of human diseases of chronic inflammatory or autoimmune character, immunoglobulin molecules display aberrant glycosylation patterns of N- or O-linked glycans. In IgA nephropathy, IgA1 molecules with incompletely galactosylated O-linked glycans in the hinge region (HR) are present in mesangial immunodeposits and in circulating immune complexes. It is not known whether the Gal deficiency in IgA1 proteins occurs randomly or preferentially at specific sites. To develop experimental approaches to address this question, the synthetic IgA1 hinge region and hinge region from a naturally Gal-deficient IgA1 myeloma protein have been analyzed by 9.4 tesla Fourier transform-ion cyclotron resonance mass spectrometry. Fourier transform-ion cyclotron resonance mass spectrometry offers two complementary fragmentation techniques for analysis of protein glycosylation by tandem mass spectrometry. Infrared multiphoton dissociation of isolated myeloma IgA1 hinge region peptides confirms the amino acid sequence of the de-glycosylated peptide and positively identifies a series of fragments differing in O-glycosylation. To localize sites of O-glycan attachment, synthetic IgA1 HR glycopeptides and HR from a naturally Gal-deficient polymeric IgA1 myeloma protein were analyzed by electron capture dissociation and activated ion-electron capture dissociation. Multiple sites of O-glycan attachment (including sites of Gal deficiency) in myeloma IgA1 HR glycoforms were identified (in all but one case uniquely). These results represent the first direct identification of multiple sites of O-glycan attachment in IgA1 hinge region by mass spectrometry, thereby enabling future characterization at the molecular level of aberrant glycosylation of IgA1 in diseases such as IgA nephropathy.     

Thiol groups and other chemical characteristics of rat monoclonal immunoglobulin A.

1. Three aspects of the monoclonal rat IgA IR22 were investigated: its free thiol groups, its hinge structure and its glycopeptides. 2. Exposed thiol groups, 1.5 per IgA dimer, were located in the Fc region, and buried thiol groups, 3.2 per IgA dimer, were located in the Fd region. 3. Rat IgA was cleaved to Fab fragments by pepsin, but, unlike mouse IgA, it was not cleaved at the hinge region by papain. 4. The contents of GlcNAc indicated that there were two N-linked glycopeptides in the Fab region, and one in the Fc or tail regions.     

Amino acid sequence requirements in the human IgA1 hinge for cleavage by streptococcal IgA1 proteases

All the IgA1 proteases of the different pathogenic species of Streptococcus cleave the hinge of the alpha chain of human IgA1 only at one proline-threonine peptide bond. In order to study the importance of these amino acids for cleavage, several hinge mutant recombinant IgA1 antibodies were constructed. The mutations were found to be without major effect upon the structure or functional abilities of the antibodies. However, they had a major effect upon their sensitivity to cleavage by some of the IgA1 proteases.     

Initiation of O-glycan synthesis in IgA1 hinge region is determined by a single enzyme,UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2

The hinge region of human immunoglobulin A1 (*IgA1) possesses multiple O-glycans, of which synthesis is initiated by the addition of GalNAc to serine or threonine through the activity of UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). We found that six pp-GalNAc-Ts, pp-GalNAc-T1, -T2, -T3, -T4, -T6, and -T9, were expressed in B cells, IgA-bearing B cells, and NCI-H929 IgA myeloma cells. pp-GalNAc-T activities of these six enzymes for a synthetic IgA hinge peptide, which has nine possible O-glycosylation sites, were examined using a reversed phase-high performance liquid chromatography, a matrix-assisted laser desorption ionization time of flight mass spectrometry, and peptide sequencing analysis. pp-GalNAc-T2 showed the strongest activity transferring GalNAc to a maximum of eight positions. Other pp-GalNAc-Ts exhibited different substrate specificities from pp-GalNAc-T2; however, their activities were extremely weak. It was reported that the IgA1 hinge region possesses a maximum of five O-glycans, and their amino acid positions have been determined. We found that pp-GalNAc-T2 selectively transferred GalNAc residues to the same five positions. These results strongly suggested that pp-GalNAc-T2 is an essential enzyme for initiation of O-linked glycosylation of the IgA1 hinge region.     

Direct evidence for decreased sialylation and galactosylation of human serum IgA1 Fc O-glycosylated hinge peptides in IgA nephropathy by mass spectrometry

Human serum immunoglobulin IgA1 is produced in bone marrow and interacts with specific cellular receptors that mediate biological events. In this study, we have analyzed the detailed glycoform structure of the human serum IgA1 Fc O-glycosylated hinge region by electrospray ionization liquid mass spectrometry. The IgA1 fragments containing the hinge glycopeptide were separated from 4 IgA nephropathy patient (IgAN) pooled sera, 10 non-IgAN pooled sera with other primary glomerulonephritides, and 5 healthy control subject pooled sera by trypsin treatment and Jacalin affinity chromatography. The molecular weights of IgA1 hinge glycopeptide were estimated using mass spectrometry, and 13 sialo and 8 asialo glycopeptide groups were identified. The results obtained clearly showed a decrease of GalNAc, Gal, and sialic acid in IgAN compared with non-IgAN and normal controls, and those strongly suggested the possibility that the decreased galactosylation and sialylation of the IgA1 hinge result in its glomerular deposition in IgAN.     

IgA1 protease

IgA1 proteases are proteolytic enzymes that cleave specific peptide bonds in the human immunoglobulin A1 (IgA1) hinge region sequence. Several species of pathogenic bacteria secrete IgA1 proteases at mucosal sites of infection to destroy the structure and function of human IgA1 thereby eliminating an important aspect of host defence. IgA1 proteases are known as autotransporter proteins as their gene structure encodes the information to direct their own secretion out of the bacterial cell. The iga gene structure is also thought to contribute to the antigenic heterogeneity demonstrated by the IgA1 proteases during infections and the cleavage specificity of the IgA1 proteases for human IgA1. The IgA1 proteases have therefore been implicated as important virulence factors that contribute to bacterial infection and colonisation. The development of strategies to inactivate these IgA1 proteases has become the subject of recent research, as this has the potential to reduce bacterial colonisation at mucosal surfaces.     

Identification of four allelic variants of the dog<Emphasis Type="Italic"> IGHA</Emphasis> gene

Multiple IgA subclasses have been identified in humans, primates and lagomorphs, whereas in mice, cattle and dogs only a single subclass has been identified. The two human subclasses (IgA₁ and IgA₂) are defined by a difference in the length of the hinge region of the chains between the CH₁ and CH₂ domains. The single IgA subclass so far identified in dogs has an -chain hinge region with a predicted amino-acid sequence similar to that of the human ₁ chain. Allelic variants that differ in the coding sequence of the hinge region have been identified in mice and pigs. In order to investigate whether allelic variants are present in dogs, a portion of the IGHA gene from eight individual dogs was cloned and sequenced. Four sequence variants were identified, and these differed in the coding region of their hinge. A major difference between the variants was the presence of a base polymorphism in the splice acceptor site for the second exon, which resulted in shortening of the hinge in two of the variants. Individuals expressed one or two of the variants identified, suggesting they may be heterozygous or homozygous. Further work is required to determine the effect of the variation on the biological activity of dog IgA and any relationship to susceptibility to mucosal disease.Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AY576788–AY576795     

Structural analyses of O-glycan sugar chains on IgA1 hinge region using SELDI-TOFMS with various lectins

The aim of the study was to develop a simple and precise method for identifying glycosylation of the IgA hinge region using surface-enhanced laser desorption/ionization (SELDI)-TOFMS with a lectin-coupled ProteinChip array. Serum IgA was isolated using an anti-IgA antibody column. Following reduction, alkylation, and trypsin digestion, the IgA fragments were applied on the ProteinChip coupled with jacalin, peanut agglutinin (PNA), or Vilsa villosa lectin (VVL). The SELDI-TOFMS peaks corresponding to the fragments containing IgA1 hinge glycopeptides trapped by each lectin were compared. The jacalin-, PNA-, and VVL-immobilized ProteinChips detected 13, 4, and 2 peaks, respectively. One major peak was confirmed as a glycopeptide by MS/MS analysis. These results suggest that a lectin-immobilized ProteinChip assay can be used to simplify the procedures for the analyses of the O-glycans in IgA1 hinge. This method potentially makes it possible to identify a disease-specific glycoform by selecting the appropriate ligand-coupled ProteinChip array.     

Endoglucanase A from Cellulomonas fimi in which the hinge sequence of human IgA1 is substituted for the linker connecting its two domains is hydrolyzed by IgA proteases from Neisseria gonorrhoeae

The hinge in IgA1 and the linker in endoglucanase A (CenA) are quite similar. The IgA1 hinge is 18 amino acids long and contains only proline, threonine and serine. The linker in CenA is 27 amino acids long and contains only proline, threonine and a single serine. IgA proteases from Neisseria gonorrhoeae cleave Pro-Ser and Pro-Thr bonds within the IgA1 hinge sequence, but they do not attack CenA. When the linker sequence of CenA is replaced with the hinge sequence of IgA1, the hybrid polypeptide is susceptible to the N. gonorrhoeae proteases. It is cleaved within the hinge sequence at the same sites as IgA1.     

IgA1 proteases from Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, and Streptococcus sanguis: comparative immunochemical studies

IgA1 proteases from H. influenzae, N. meningitidis, S. pneumoniae, and S. sanguis were compared with respect to site of cleavage in the IgA1 molecule and EDTA sensitivity. Proteases from S. sanguis and S. pneumoniae cleaved the Pro (227)-Thr (228) bond within the hinge region of the alpha 1 chain and were inhibited by EDTA. H. influenzae IgA1 protease cleaved the Pro (231)-Ser (232) peptide bond. The activity of IgA1 proteases from H. influenzae and N. meningitidis was unaffected by EDTA. Purified and denatured alpha 1 chain was cleaved only in the hinge region. Other component chains of secretory IgA (secretory component, light and J chains) were not susceptible. In addition to IgA1 protease, S. pneumoniae released exo- and endoglycosidases that removed a considerable portion of carbohydrate side chains of IgA1; this activity was absent from crude IgA1 protease preparations of the other three bacterial species. Association in vitro of polymeric IgA1 with SC did not inhibit the degradation of IgA1 proteases. The considerable resistance of secretory IgA to cleavage by IgA1 proteases may be explained in part by the presence of IgA1 protease-neutralizing antibodies in secretory IgA.     

Protein dynamics in the region of the sixth ligand methionine revealed by studies of imidazole binding to Rhodobacter capsulatus cytochrome c2 hinge mutants

All class I c-type cytochromes studied to date undergo a dynamic process in the oxidized state, which results in the transient breaking of the iron-methionine-sulfur bond and sufficient movement to allow the binding of exogenous ligands (imidazole in this work). In the case of Rhodobacter capsulatus cytochrome c(2), the sixth heme ligand Met96 and up to 14 flanking residues (positions 88-100, termed the hinge region), located between two relatively rigid helical regions, may be involved in structural changes leading to a transient high-spin species able to bind ligands. We have examined 14 mutations at 9 positions in the hinge region of Rhodobacter capsulatus cytochrome c(2) and have determined the structure of the G95E mutant. Mutations near the N- and C-terminus of the hinge region do not affect the kinetics of movement but allow us to further define that portion of the hinge that moves away from the heme to the 93-100 region in the amino acid sequence. Mutations at positions 93 and 95 can alter the rate constant for hinge movement (up to 20-fold), presumably as a result of altering the structure of the native cytochrome to favor a more open conformation. The structure of one of these mutants, G95E, suggests that interactions within the hinge region are stabilized while interaction between the hinge and the heme are destabilized. In contrast, mutations at positions 98 and 99 alter imidazole binding kinetics but not the hinge movement. Thus, it appears that these mutations affect the structure of the cytochrome after the hinge region has moved away from the heme, resulting in increased solvent access to the bound imidazole or alter interactions between the protein and the bound imidazole.     

The Fab and Fc fragments of IgA1 exhibit a different arrangement from that in IgG: a study by X-ray and neutron solution scattering and homology modelling

Human immunoglobulin A (IgA) is an abundant antibody that mediates immune protection at mucosal surfaces as well as in plasma. The IgA1 isotype contains two four-domain Fab fragments and a four-domain Fc fragment analogous to that in immunoglobulin G (IgG), linked by a glycosylated hinge region made up of 23 amino acid residues from each of the heavy chains. IgA1 also has two 18 residue tailpieces at the C terminus of each heavy chain in the Fc fragment. X-ray scattering using H2O buffers and neutron scattering using 100 % 2H2O buffers were performed on monomeric IgA1 and a recombinant IgA1 that lacks the tailpiece (PTerm455). The radii of gyration RG from Guinier analyses were similar at 6.11-6.20 nm for IgA1 and 5.84-6.16 nm for PTerm455, and their cross-sectional radii of gyration RXS were also similar. The similarity of the RG and RXS values suggests that the tailpiece of IgA1 is not extended outwards in solution. The IgA1 RG values are higher than those for IgG, and the distance distribution function P(r) showed two distinct peaks, whereas a single peak was observed for IgG. Both results show that the hinge of IgA1 results in an extended Fab and Fc arrangement that is different from that in IgG. Automated curve-fit searches constrained by homology models for the Fab and Fc fragments were used to model the experimental IgA1 scattering curves. A translational search to optimise the relative arrangement of the Fab and Fc fragments held in a fixed orientation resembling that in IgG was not successful in fitting the scattering data. A new molecular dynamics curve-fit search method generated IgA1 hinge structures to which the Fab and Fc fragments could be connected in any orientation. A search based on these identified a limited family of IgA1 structures that gave good curve fits to the experimental data. These contained extended hinges of length about 7 nm that positioned the Fab-to-Fab centre-to-centre separation 17 nm apart while keeping the corresponding Fab-to-Fc separation at 9 nm. The resulting extended T-shaped IgA1 structures are distinct from IgG structures previously determined by scattering and crystallography which have Fab-to-Fab and Fab-to-Fc centre-to-centre separations of 7-9 nm and 6-8 nm, respectively. It was concluded that the IgA1 hinge is structurally distinct from that in IgG, and this results in a markedly different antibody structure that may account for a unique immune role of monomeric IgA1 in plasma and mucosa.     

Mutations at the interdomain hinge region of the DadB alanine racemase: effects of length and conformational constraint of the linker sequence on catalytic efficiency

The pentapeptide "hinge" region of the DadB alanine racemase links two structural domains of the protein [Galakatos, N. G., & Walsh, C. T. (1987) Biochemistry 26, 8475]. The presence of substrate markedly reduces the rate of hinge-specific proteolysis of this racemase and induces a conformational change observed by circular dichroism. To evaluate the possible contribution of the proteolytically sensitive hinge residues (-Y253GGGY257-) on catalytic efficiency, site-directed mutations were generated to probe the effects of size and conformational rigidity of that region. A bacterial overproducing system for the dadB gene was constructed that expresses the enzyme as 4.5% of total soluble protein. On this construct, a four-part ligation allowed the engineering of two unique and proximal restriction sites required for cassette mutagenesis at the hinge region. For two of the eight mutants generated, expressed protein could not be detected (deletion of -GGGY-; termination codon at position 255). Deletion of one or two of the three Gly residues had no effect on catalytic efficiency. Insertion of a fourth Gly resulted in a 5-fold drop in Vmax/Km. For G254P, G255P, and G256P, Vmax/Km was 60%, 126%, and 26% of the native enzyme, respectively. In all cases, the Km's remained essentially constant, suggesting that the hinge region is not involved in substrate binding. The rate of hinge-specific proteolysis of the mutants was faster than that of wild-type DadB except for the G255P protein for which it was equivalent.     

Immunoglobulin genetics of Ornithorhynchus anatinus (platypus) and Tachyglossus aculeatus (short-beaked echidna)

In this paper, we review data on the monotreme immune system focusing on the characterisation of lymphoid tissue and of antibody responses, as well the recent cloning of immunoglobulin genes. It is now known that monotremes utilise immunoglobulin isotypes that are structurally identical to those found in marsupials and eutherians, but which differ to those found in birds and reptiles. Monotremes utilise IgM, IgG, IgA and IgE. They do not use IgY. Their IgG and IgA constant regions contain three domains plus a hinge region. Preliminary analysis of monotreme heavy chain variable region diversity suggests that the platypus primarily uses a single VH clan, while the short-beaked echidna utilises at least 4 distinct VH families which segregate into all three mammalian VH clans. Phylogenetic analysis of the immunoglobulin heavy chain constant region gene sequences provides strong support for the Theria hypothesis. The constant region of IgM has proven to be a useful marker for estimating the time of divergence of mammalian lineages.     

Genetically engineered polymers of human CuZn superoxide dismutase. Biochemistry and serum half-lives

CuZn superoxide dismutase is a highly stable dimer of identical subunits with a combined molecular mass of 32,000 daltons. Two human superoxide dismutase genes have been joined in the same translational reading frame, using spacers of different lengths, to encode single chain proteins consisting of two identical human superoxide dismutase subunits. The first construct encodes two directly linked subunits; the terminal glutamine codon of the first gene was changed to a methionine codon and followed immediately by the second gene. The second construct encodes two subunits linked by a 19-amino-acid human immunoglobulin IgA1 hinge sequence. Both constructs produce high levels of catalytically active superoxide dismutase when expressed in Escherichia coli. The protein containing the IgA1 hinge sequence forms polymers up to 750,000 in molecular weight, which are linked together noncovalently by the hydrophobic bonding of the dimer interface. The polymers are soluble, thermostable, and of near normal specific activity. Site-directed in vitro mutagenesis was used to inactivate one of the two human superoxide dismutase subunits. The resulting human superoxide dismutase polymers have approximately 50% activity, thus confirming that the products of both genes are catalytically active. Large amounts of individual polymeric forms have been purified from recombinant yeast and tested for serum stability in rats. The serum half-life is approximately 7 min for both the two-chain wild type human superoxide dismutase dimer (Mr 32,000) and the single chain molecule consisting of a human superoxide dismutase dimer covalently linked by the immunoglobulin hinge region (Mr 34,000), whereas the higher molecular weight polymers (Mr greater than or equal to 68,000) all have half-lives of approximately 145 min.     

Mouse IgA allotypes have major differences in their hinge regions

Six IgA allotypes are serologically identifiable in inbred mice. The sequences of the PCR-amplified C alpha 1, C alpha 2 and C alpha 3 exons from the genomic DNA of mice of four previously unsequenced allotypes already have been compared with those of BALB/c and of a wild mouse, Mus pahari, in the literature. Sporadic differences, including several that may encode the known allotypic determinants, are found throughout the three exons, but major differences occur in the hinge. The hinge is longest in DBA/2 ( Igh-2(c)) mice, having an extra codon compared with that of BALB/c ( Igh-2(a)) and B10.A ( Igh-2(b)) mice. It is two codons shorter in CE ( Igh-2(f)) and four shorter in M. pahari, AKR and NZB (both Igh-2(d)) mice, but the position of the missing codons in the latter two strains is offset from that in M. pahari. The hinges in BALB/c ( Igh-2(a)) and DBA/2 ( Igh-2(c)) differ most from each other and from the other three allotypes, which are fairly closely related. Both BALB/c and DBA/2 have O-linked glycosylation sites, but they are in different positions in the hinge. Compared with BALB/c ( Igh-2(a)), B10.A( Igh-2(b)) has two extra Cys residues in the hinge, while DBA/2 ( Igh-2(c)), AKR/NZB ( Igh-2(d)) and CE ( Igh-2(f)) each have one. The differences in hinge length may have arisen by mismatching of highly repetitive portions of its sequence during meiotic recombination. Possible effects of the differences in hinge length and composition on the behavior of the mouse IgA allotypes are discussed.     

The influences of hinge length and composition on the susceptibility of human IgA to cleavage by diverse bacterial IgA1 proteases

The influences of IgA hinge length and composition on its susceptibility to cleavage by bacterial IgA1 proteases were examined using a panel of IgA hinge mutants. The IgA1 proteases of Streptococcus pneumoniae, Streptococcus sanguis strains SK4 and SK49, Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae cleaved IgA2-IgA1 half hinge, an Ab featuring half of the IgA1 hinge incorporated into the equivalent site in IgA1 protease-resistant IgA2, whereas those of Streptococcus mitis, Streptococcus oralis, and S. sanguis strain SK1 did not. Hinge length reduction by removal of two of the four C-terminal proline residues rendered IgA2-IgA1 half hinge resistant to all streptococcal IgA1 metalloproteinases but it remained sensitive to cleavage by the serine-type IgA1 proteases of Neisseria and Haemophilus spp. The four C-terminal proline residues could be substituted by alanine residues or transferred to the N-terminal extremity of the hinge without affect on the susceptibility of the Ab to cleavage by serine-type IgA1 proteases. However, their removal rendered the Ab resistant to cleavage by all the IgA1 proteases. We conclude that the serine-type IgA1 proteases of Neisseria and Haemophilus require the Fab and Fc regions to be separated by at least ten (or in the case of N. gonorrhoeae type I protease, nine) amino acids between Val(222) and Cys(241) (IgA1 numbering) for efficient access and cleavage. By contrast, the streptococcal IgA1 metalloproteinases require 12 or more appropriate amino acids between the Fab and Fc to maintain a minimum critical distance between the scissile bond and the start of the Fc.     

Analysis of the microheterogeneity of the IgA1 hinge glycopeptide having multiple O-linked oligosaccharides by capillary electrophoresis

It was found that the self-aggregation of IgA1 was closely connected with the glycoform of a mucin-type sugar chain on its hinge portion. In this report, normal human serum IgA1 was separated into two subfractions by a jacalin column. The elution condition, 25 mM galactose, used here was similar to that reported for the glycoprotein with a single mucin-type sugar chain per molecule. The IgA1 eluted under this condition was substantially the monomeric form. In contrast, the remaining IgA1 eluted from the column with 0. 8 M galactose was substantially the aggregated form. An analytical method for the microheterogeneity of the IgA1 hinge glycopeptide (HGP33) was developed to determine the difference between these IgA1 fractions by capillary electrophoresis (CE). Native HGP33 from both IgA1 fractions was separated into peaks 1-11, depending on their glycoforms. Because the sialic acid-rich component migrated slowly on CE, the 25 mM fraction was abundant in the sialic acid-rich components (peaks 7-11), but the 0.8 M fraction was abundant in the sialic acid-poor components (peaks 1-4). Comparison of the number of sugar chains per hinge peptide indicated that the 25 mM fraction was relatively well glycosylated. Thus, application of CE analysis to the HGP33 indicated that the monomeric IgA1 was composed of a relatively complete molecule with respect to the glycoform rather than the aggregated IgA1.     

Evolutionary hypervariability in the hinge region of the immunoglobulin alpha gene

The hinge region of the immunoglobulin molecule is responsible for antigen-binding and cross-linking reactions, varying the distance between the two antigen-binding sites. As the amino acid sequence of the hinge region is identical among immunoglobulin molecules of the same (sub)class, it has been regarded as a constant region. By comparison of the nucleotide sequences among primate C alpha genes, it is clear that there is a wide variety of length among the hinge regions of hominoid C alpha genes, which basically consist of tandem repeats of a 15 base-pair sequence. This reiterated structure probably facilitates rapid evolutionary changes in the length of the hinge region. The hinge region of the Old World monkey C alpha gene has a non-reiterated structure whose nucleotide sequence is quite different from those of the hominoid C alpha genes, although its surrounding region is conserved during evolution. This unusual hypervariability reveals that the hinge region has evolved as a semi-variable region in contrast to its constant character from an ontogenic viewpoint.