Recombination of two homologous MHC class III genes of the mouse (C4 and Slp) that accounts for the loss of testosterone dependence of sex-limited protein expression

In most mouse strains, expression of a gene encoding sex-limited protein (Slp), an isotype of the fourth component of complement (C4), is induced by testosterone, or the gene is not expressed at all; however, in some wild-derived strains carrying H-2w7, H-2w16, or H-2w19 haplotype, Slp is expressed constitutively in the same way as C4. To examine the structural basis for the testosterone-independent expression of Slp, 41 overlapping clones together encoding the S region were isolated from C3H.W7 mouse (H-2w7) cosmid library. Five C4-related genes each spanning approximately 16 kb were identified among the cluster of cosmid clones and were isolated for structural study. One of the genes (C4w7) hybridized with the C4-specific oligonucleotide probe but not with the Slp-specific oligonucleotide probe, whereas the other genes (Slpw7a, Slpw7b, Slpw7c, and Slpw7d) hybridized only with the Slp-specific probe. Restriction mapping of these genes and sequencing of the selected regions of 5'-flanking regions of the genes were performed, and the results were compared with the data obtained with the C4 and Slp genes of FM (H-2d) and B10.BR (H-2k). These studies showed that three of the C4-related genes of C3H.W7 (Slpw7b, Slpw7c, and Slpw7d) are C4-Slp recombinant genes comprising a 5'-region derived from C4 gene and a 3'-region derived from Slp gene. It is suggested that 5'-flanking region derived from C4 in these C4-Slp recombinant genes accounts for testosterone-independent expression of Slp in C3H.W7 mouse.     

Variation in the glycosylation of the murine sex-limited protein: comparison with the fourth component of murine complement

The murine sex-limited protein (Slp) is a hemolytically nonfunctional homologue of the fourth component of complement (C4). Two congenic mouse strains, B10.BUA1 (H-2w16) and B10.KPB128 (H-2w19), which have been previously shown to share a variant form of C4 (Karp et al., J. Biol. Chem., 257: 7330-7335), were examined and found to also produce a variant form of Slp. Slp molecules isolated from the plasma or peritoneal macrophage cultures from these strains have an alpha-chain approximately 2,000 daltons smaller than the alpha-chain of Slp from H-2d or H-2w7 mice as judged by sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis. Expression of this Slp was constitutive, i.e., not regulated by androgen, and is cis-dominant in F1 hybrid mice. Autolysis of the different relative molecular mass (Mr) alpha-chains at the internal thiolester produced similar Mr amino-terminal fragments and different Mr carboxy-terminal fragments. Deglycosylation of the alpha-chains with trifluoromethanesulfonic acid eliminated most, if not all, the Mr difference. The Mr difference was also manifested by the intracellular precursors of Slp and could be eliminated by endoglycosidase H (endo H) treatment. The number of oligosaccharides on the Slp alpha-chain was deduced by limited endo H treatment of Slp synthesized in the presence of swainsonine, a plant alkaloid that prevents maturation of complex-type oligosaccharides. This method is a simple way to enumerate the complex-type, N-linked oligosaccharides on glycoproteins. The genetic variation in the glycosylation of Slp was compared with the known variation in glycosylation of C4, and a scheme depicting some of the structural differences among these molecules was developed.     

Constitutive expression of Slp genes in mouse strain B10.WR directed by C4 regulatory sequences

The murine fourth component of complement (C4) and sex-limited protein (Slp) are two closely related serum proteins that exhibit very disparate patterns of gene expression: all mice constitutively express C4, whereas only adult male mice from a limited number of standard inbred strains express Slp. Several exceptional strains exhibit constitutive (C4-like) Slp expression, a phenotype that correlates with multiple copies of the Slp gene. To determine the molecular basis for constitutive Slp expression we have isolated genomic clones and compared the sequences of 1.5 kb of 5' flanking DNA from 1 C4 gene and three different Slp genes from the Slp-constitutive strain B10.WR. These sequence comparisons demonstrate C4-like regulatory sequences adjacent to two of the Slp genes. By analysis of cDNA clones isolated from a B10.WR liver library we demonstrate that the constitutive Slp phenotype is due primarily to expression of one of these C4/Slp hybrid genes. It appears likely that Slp gene duplication in strain B10.WR came about via homologous unequal crossover events between C4 and Slp genes; this would accommodate both the gene sequence data and the pattern of C4-like Slp expression in mouse strain B10.WR.     

The murine Slp gene. Additional evidence that sex-limited protein has no biologic function

Sex-limited protein (Slp) is a mouse serum protein of unknown function that has approximately 95% amino acid sequence identity with murine complement component C4 but is inactive in the complement pathway. The gene for Slp lies in the S region of the murine H-2 complex adjacent to the gene Cyp21 that encodes the Cytochrome P-450 enzyme steroid 21-hydroxylase. We report the sequence of a 26,307 bp long segment of the mouse genome that includes both the Slp and Cyp21 genes. The sequence reported was assembled from the sequences of three overlapping lambda phage genomic clones from mouse strain B10.WR, which carries four tandem pairs of Slp and Cyp21 genes. We also report the sequence of a fourth lambda clone, 12,539 bp in length, carrying parts of a distinct pair of Slp and Cyp21 genes from B10.WR mice. The Slp gene at 14.3 kb in length is about 1 kb shorter than the C4 gene; this difference is due primarily to absences of a simple repetitive sequence and a middle repetitive MT element in the corresponding introns 14 and 15, respectively. The gene sequence reveals an intron/exon organization identical to that of the murine C4 gene, and also that the 9 nucleotide deletion in exon 18, which appears to be directly responsible for the absence of complement activity, is unrelated to differences in intron sequences. Detailed comparisons of C4 and Slp gene sequences indicate that nucleotide substitutions in the Slp gene are occurring at approximately the same rate in both exons and introns. This implies that the murine Slp gene resembles a pseudogene and supports previously reported evidence that the Slp protein has no biologic function.     

Mice constitutive for sex-limited protein (SLP) expression contain multiple Slp gene sequences

The murine fourth component of complement (C4) and sex-limited protein (Slp) are two closely related serum proteins whose structural genes map to the S region of H-2. Serum C4 levels vary as much as 20-fold between C4 high (C4H) and C4 low (C4L) strains, and Slp expression can be null (SlpO), limited to male mice of a subset of C4H strains (Slp+), or "constitutive" (SlpC), in which female as well as male mice express Slp. In this study, we compare, by genomic Southern blot analysis, the C4 and Slp genes from eight congenic inbred mouse strains representative of three distinct phenotypes: C4H, Slp+ (two strains), C4H, SlpO (two strains), C4H, SlpC (three strains), and C4L, SlpO (one strain). By using cDNA probes that recognize both C4 and Slp genes, and are derived from the extreme 5' and 3' ends of the mRNA as well as internal coding sequences, we find no evidence to suggest that strain-specific variations in the expression of C4 and Slp are due to gross deletions of major portions of the structural genes. In most cases, two distinct C4/Slp genes are detected; hybridization with C4- and Slp-specific probes indicate that one of these is C4 and the other is Slp. The three SlpC strains are exceptional: they carry at least four C4/Slp genes; one of these hybridizes to the C4-specific probe whereas the remaining genes hybridize to the Slp-specific probe. Hence, multiple duplication of a gene containing Slp sequences has occurred in certain strains of mice, and this is accompanied by constitutive expression of the Slp protein.     

Restriction fragment length polymorphism of the murine C4 and Slp genes: two C4 groups.

We have examined the related H-2 genes coding for the fourth component of complement (C4) and the sex-limited protein (Slp) from 30 inbred mouse strains by Southern blot analysis. With four restriction enzymes, 11 RFLP patterns distributed among 26 different H-2 haplotypes have been identified. Strains of the same serologic H-2 haplotype were found to have identical RFLP patterns. It was confirmed that the number of C4-related genes in most haplotypes is two, Slp and C4; but H-2SWI6 (SWI6) and SWI9, which have the same RFLP pattern, have four and Sw7 has five. Although C4 and Slp have many similarities, they also were found to contain distinctive features: relative to Slp, each C4 allele examined has two insertions totaling 1.1 kb located in introns 14 and 15; and each Slp allele examined, excluding hybrids, has a provirus insertion upstream. No other large deletions or insertions were detected. The RFLP patterns are also due to 10 polymorphic restriction sites, which have been placed on standard maps; two are associated with Slp and eight are associated with C4.Sk strains, the only strains that express low serum levels of C4, have the same RFLP phenotype as Sw14, Sw18, and Swx; Sk may have arisen from a recent common ancestor of these strains. Homologous recombination has been important in the formation of existing C4 alleles. However, based on complete linkage disequilibrium between three RFLP internal to C4, the haplotypes have been divided into two groups that may have functional significance.     

Aberrant splicing caused by the insertion of the B2 sequence into an intron of the complement C4 gene is the basis for low C4 production in H-2k mice.

The serum level of the fourth component of complement (C4) in mice bearing the H-2k haplotype is only 1/10 to 1/20 of that of non-H-2k mice. We have analyzed C4 cDNA clones from B10.BR(H-2k) mouse liver and found aberrant C4 cDNA which contained a 200-base pair (bp) insertion between the exon 13 and exon 14 encoded sequences in addition to the normal C4 cDNA. The 5' 148 bp and the 3' 52 bp of this insert were derived from the B2 sequence, the short interspersed repeats of mouse genome, and the central part of intron 13, respectively. Sequence analysis of intron 13 of the C4k gene showed the presence of a complete copy of a B2 consensus sequence. The structure of aberrant C4 mRNA indicated that the possible 3' splice site in the B2 sequence and the cryptic 5' splice site in intron 13 were used. Both the insertion of the B2 sequence into intron 13 and the presence of aberrant mRNA in the liver were specific to H-2k-bearing mice, suggesting that the aberrant splicing due to the B2 insertion is the basis for low C4 expression in H-2k mice.     

Complete nucleotide and derived amino acid sequences of sex-limited protein (Slp), nonfunctional isotype of the fourth component of mouse complement (C4)

The nucleotide sequence coding for sex-limited protein (Slp), the testosterone-regulated isotype of the fourth component of mouse complement (C4), has been determined from cloned genomic DNA and cDNA fragments. The complete deduced amino acid sequence for the single chain precursor protein of Slp (pro-Slp) consists of 1716 residues. The mature beta, alpha, and gamma subunits contain 654, 763, and 291 amino acids, respectively. One potential carbohydrate attachment site is predicted from the beta-chain, five for the alpha-chain, and none for the gamma-chain. From the comparison with the mouse C4 sequences, an extensive overall sequence homology, 96.0% in nucleotides and 94.2% in amino acids, is observed. Only one deletion/insertion event is recognized between C4 and Slp sequences: three residues near the Cls cleavage site are deleted from Slp. The distribution of cysteine residues is completely conserved between pro-Slp and pro-C4.     

Murine sex-limited protein (Slp) antigenic sites in human complement component C4

Human complement component C4 is encoded by two HLA-linked loci, A and B. In the mouse, the H-2 region contains structural genes for two serum proteins that react with antibodies to human C4, but one of these proteins (Slp) has no C4 hemolytic activity. Because the product of C4-A locus in man has low hemolytic activity, a previous report suggests it may be the homologue of murine Slp. We show here that Slp antigenic determinants are found in human C4. However, they are expressed in the products of both loci A and B, that is, C4A and C4B, since both proteins were specifically immunoprecipitated by the IgG fraction of alloantisera to mouse Slp. Therefore, Slp-associated structural features are preserved in evolution, although they do not seem to be relevant to the hemolytic properties of C4.     

Murine sex-limited protein: complete cDNA sequence and comparison with murine fourth complement component

Murine sex-limited protein (Slp) is a structural homologue of the murine fourth complement component (C4) that lacks C4 activity and has no known function. The genes for C4 and Slp lie closely linked in the S region of the murine major histocompatibility complex. We have sequenced a cDNA clone that spans the entire protein-coding region of Slp from the mouse strain B10.WR. The sequence contains a 1735 amino acid-long open reading frame encoding a putative prepro-Slp flanked by 51 and 103 untranslated nucleotides at the 5' and 3' ends respectively; it shows 96% nucleotide and 94% amino acid identity with our previously reported complete sequence of murine C4 from the same mouse strain. The present complete Slp sequence differs slightly from our previously reported partial sequence from the same mouse strain; this suggests that at least two distinct Slp genes are transcribed in B10.WR mice. We suggest, by analogy with procaryotic DNA-binding proteins, that a three amino acid deletion in Slp, close to the Cls cleavage site, makes that site resistant to proteolysis; this renders Slp inactive. We also speculate on the possibility that Slp might be a gene in evolutionary transition; one that is midway in the evolution of a completely silent pseudogene or a new gene with a novel function.     

Phylogenetic conservation of a class III major histocompatibility complex antigen, factor B. Isolation and nucleotide sequencing of mouse factor B cDNA clones

The complement protein factor B is a novel serine protease which is encoded within the major histocompatibility complex in man, guinea pig, and mouse. To determine the structure of mouse factor B, cDNA clones were isolated from mouse strains of two different major histocompatibility complex haplotypes, H-2k and H-2d, and clones of 0.9 and 1.5 kilobases, respectively, were sequenced. The H-2d clone includes the H-2k clone sequence and spans 94% of the Bb-coding sequence. No differences in sequence or in restriction enzyme sites were observed between the H-2k and H-2d clones. The H-2d clone displays 83% nucleotide homology and 83% (derived) amino acid homology with that of human factor B; there are no insertions or deletions. Comparison of the mouse and human factor B sequence reveals extensive regional homology at the catalytic residues and in the NH2-terminal portion of the Bb fragment.     

Insertion of the B2 sequence into intron 13 is the only defect of the H-2k C4 gene which causes low C4 production.

The serum level of the fourth component of complement (C4) in mice bearing H-2k haplotype is only 1/10 of that of non-H-2k mice. H-2k bearing mice, but not non-H-2k bearing mice, have an insertion of the B2 sequence into intron 13 of the C4 gene, and aberrant C4 mRNA in liver apparently generated by abnormal RNA splicing caused by the insertion of the B2 sequence. To test the possible causal relationship between the B2 insertion and low C4 production in H-2k mice directly, we constructed the H-2k C4 gene without the B2 insertion and the H-2w7 (non-H-2k) C4 gene with the B2 insertion by exchanging a part of intron 13 between these two genes. Transfection of the intact H-2w7 C4 gene or the chimeric H-2k gene without the B2 insertion into HepG2 cells resulted in the production of only normal C4 mRNA at the normal level. On the other hand, the intact H-2k C4 gene or the chimeric H-2w7 C4 gene with the B2 insertion directed production of both aberrant and a decreased amount of normal C4 mRNA. These results demonstrated that the insertion of B2 sequence into intron 13 of the C4 gene is the only determinant of low C4 production by H-2k mice through aberrant RNA processing.     

The C4 and Slp genes of the complement region of the murine H-2 major histocompatibility complex

Recent analyses, at the protein and DNA levels of structure, of the murine complement components C4 and the closely related sex-limited protein, Slp have led to new insights into the H-2/S region-linked C4 and Slp genes and their products. The primary products are 200 000 Da precursors which are cleaved, intracellularly and extracellularly, into the the mature alpha-beta-gamma-subunit molecules of plasma. Precursor order of subunits is beta-alpha-gamma; a complementary DNA clone spanning the alpha-gamma junction has been extensively analysed. The C-terminal of the alpha-chain is of particular interest because of post-secretion processing which differentiates 'secreted' and 'plasma' forms of C4, both apparently functional, and because allelic variants of C4 and the Slp protein, which differ substantially in molecular masses, owe their differences principally to different levels of glycosylation of the alpha-chain. Allelic variations in rate of C4 synthesis (C4-high compared with C4-low) have been analysed in cultures of hepatocytes and macrophages. Three distinct modes of genetic regulation of the expression of the Slp protein have been identified.     

Trans-regulatory genes affect Slpa and Slpo expression and act in a tissue-specific manner

The plasma protein C4 and its androgen-dependent homologue Slp are encoded by genes located in the mouse major histocompatibility complex, H-2. The C4 and Slp protein levels and liver mRNA levels are influenced by non-H-2-linked regulatory genes. The allele-specific regulation of C4 expression and the androgen regulation of Slp expression are manifest only in some of the tissues where these genes are expressed. Therefore, we studied tissues in which the effects of the non-H-2 regulatory genes are apparent. We show that these genes only affect the Slp expression in tissues where it is androgen-dependent. This indicates that the non-H-2 regulatory genes most likely act through interaction with the androgen regulation of Slp expression. We also show that liver cells of mice with the Slp ^o allele, which do not produce Slp protein, do express Slp mRNA; this expression is also androgen-induced and regulated by non-H-2 genes. Thus, both the Slp ^a and Slp ^o alleles appear to be regulated in the same way.     

Molecular regulation of MHC class III (C4 and factor B) gene expression in mouse peritoneal macrophages

To study the molecular regulation of C4 and factor B synthesis in mouse peritoneal macrophages, mouse C4 cDNA clones isolated from an H-2d haplotype liver cDNA library, and a previously described mouse factor B cDNA clone, pBmB2 (9), were used to assess quantitative and qualitative differences in C4 and factor B mRNA in resident and elicited cells. The C4 clones that were isolated, pBmS2 (1 Kb) and pBmS10 (0.9 Kb), overlap and together span a 1.5 Kb coding region of mouse pro-C4, extending from the alpha-chain through the gamma-chain; four nucleotide substitutions are evident in comparing 316 bp of the sequence of clone pBmS10 to that of a previously described mouse C4 clone, pMLC4/w7-2 (23). By using these probes, Northern blot analysis of total cellular RNA revealed similar C4 mRNA levels in resident peritoneal macrophages from high-C4 (B10.A) and low-C4 (C3HeB) strains. Pulse and pulse-chase studies of C4 and factor B synthesis were performed on resident, starch-elicited, and thioglycollate-elicited peritoneal macrophages at two culture time periods, 0 to 9 and 24 to 33 hr, and total cellular RNA was isolated from each population at 4.5 and 28.5 hr of culture for Northern blot analysis of C4 and factor B mRNA content. The data demonstrate that as previously reported, C4 production decreases in elicited compared with resident macrophages and decreases with time in culture; however, factor B synthesis does not differ among resident and elicited cells and it increases with time in culture. The variations in C4 and factor B production by mouse peritoneal macrophages are not associated with alterations in C4 and factor B protein processing, catabolism, or secretion; rather, they are a function of differences in net amounts of C4 and factor B mRNA. These data provide direct evidence that the regulation of expression of these class III MHC genes in mouse peritoneal macrophages is a pretranslational event.     

Regulation of expression of mouseC4 andSlp genes by non-H-2-linked genes

C4 (the fourth complement component) and Slp (sexlimited protein) are two homologous plasma proteins encoded by genes in theS-region of theH-2 gene complex. We studied the genetic factors influencing the plasma levels of these proteins and their mRNA levels in liver. Considerable differences in both protein and mRNA levels were found between mouse strains carrying the sameS-region allele on different genetic backgrounds, indicating a pretranslational effect of non-H-2-linked genes on the expression of the twoS-region genes. The expression of Slp is androgen-dependent in the strains tested. However, testosterone treatment cannot increase the low levels of Slp caused by non-H-2-linked regulatory genes. In mice with Slp-negativeS-region alleles we found liver mRNA hybridizing with Slp-specific oligonucleotides, indicating expression of theSlp gene in Slp-negative strains. Our data demonstrate the complexity of the regulation of theC4 andSlp genes and pave the way for the analysis of the regulatory factors involved.