Disulfide bond rearrangement during formation of the chorionic gonadotropin beta-subunit cystine knot in vivo

The intracellular kinetic folding pathway of the human chorionic gonadotropin beta-subunit (hCG-beta) reveals the presence of a disulfide between Cys residues 38-57 that is not detected by X-ray analysis of secreted hCG-beta. This led us to propose that disulfide rearrangement is an essential feature of cystine knot formation during CG-beta folding. To test this, we used disulfide bond formation to monitor progression of intracellular folding intermediates of a previously uncharacterized protein, the CG-beta subunit of cynomolgous macaque (Macaca fascicularis). Like its human counterpart hCG-beta with which it shares 81% identity, macaque (m)CG-beta is a cystine knot-containing subunit that assembles with an alpha-subunit common to all glycoprotein hormone members of its species to form a biologically active heterodimer, mCG, which, like hCG, is required for pregnancy maintenance. An early mCG-beta folding intermediate, mpbeta1, contained two disulfide bonds, one between Cys34 and Cys88 and the other between Cys38 and Cys57. The subsequent folding intermediate, mpbeta2-early, was represented by an ensemble of folding forms that, in addition to the two disulfides mentioned above, included disulfide linkages between Cys9 and Cys57 and between Cys38 and Cys90. These latter two disulfides are those contained within the beta-subunit cystine knot and reveal that a disulfide exchange occurred during the mpbeta2-early folding step leading to formation of the mCG-beta knot. Thus, while defining the intracellular kinetic protein folding pathway of a monkey homologue of CG-beta, we detected the previously predicted disulfide exchange event crucial for CG-beta cystine knot formation and attainment of CG-beta assembly competence.     

A novel four-amino acid determinant defines conformational freedom within chorionic gonadotropin beta-subunits

On the basis of apparent molecular mass heterogeneity following reducing versus nonreducing SDS-PAGE, we determined that the beta-subunit of macaque (Macaca fascicularis) chorionic gonadotropin (mCG-beta) is more conformationally constrained than the beta-subunit of human chorionic gonadotropin (hCG-beta). The amino acid sequences of these two subunits are 81% identical. To determine the conformational variance source, which was not due to glycosylation differences, we generated a series of hCG-beta-mCG-beta chimeras and identified domains that contributed to CG-beta conformational freedom. We discovered that the CG-beta 54-101 domain contained a small subdomain, residues 74-77, that regulated the conformational freedom of the beta-subunit; i.e., when residues 74-77 were of macaque origin (PGVD), the mutated hCG-beta subunit displayed macaque-like conformational rigidity, and when residues 74-77 were of human origin (RGVN), the mutated mCG-beta subunit displayed human-like conformational freedom and microheterogeneity. Additionally, CG-beta N-terminal domain residues (8, 18, 42, and 46-48) were also found to influence CG-beta conformational freedom when residues 74-77 were of human but not macaque origin. The biological significance of the CG-beta conformational variance was tested using a biological assay that showed that the hCG-alpha-hCG-beta heterodimer facilitated human CG receptor-mediated cAMP-driven luciferase reporter gene activity in HEK cells nearly 1 order of magnitude more effectively than the hCG-alpha-mCG-beta chimera. Together, these data demonstrate that two essential amino acid residues within a four-amino acid subdomain regulated CG-beta conformational freedom and that a conformational difference between hCG-beta and mCG-beta was recapitulated in the context of receptor-mediated CG heterodimer signal transduction activation.     

A new subclass of the luteinizing hormone/chorionic gonadotropin receptor lacking exon 10 messenger RNA in the New World monkey (Platyrrhini) lineage

The luteinizing hormone receptor (LHR) plays an essential role as a mediator of LH and CG action during embryonic sexual differentiation and in gametogenesis. In a hypogonadal male patient, we recently demonstrated that a genomic deletion of exon 10, located in the hinge region of the extracellular domain, results in discrimination of LH and hCG action. In the common marmoset (Calltithrix jacchus), exon 10 of the LHR is naturally missing at the mRNA level. In order to investigate whether this is an isolated species-specific phenomenon, we performed a phylogenetic screening, searching for the presence of LHR exon 10 mRNA in a number of primate species representative for the major lineages of primate evolution. The expressed LHR region encompassing exon 10 was amplified from testicular tissue by RT-PCR, cloned, and sequenced. In addition, we performed Southern blot analysis of the LHR of selected New World and Old World primates. The results revealed that exon 10 mRNA is lacking in the complete New World monkey (Platyrrhini) lineage but is present in both more primitive and more advanced primates. However, exon 10 seems to be present at the genomic level, arguing for a splicing failure possibly due to a genomic mutation or the lack of appropriate splicing factors. Considering that, in the human, LH is far less active than hCG on the LHR lacking exon 10, we addressed the question whether the existence of such a receptor has any consequences on the dual hormone LH/CG system present in Platyrrhini. Using primers specific for the known marmoset CG beta cDNA, we amplified the CG beta subunit cDNA from male common marmoset pituitaries by RT-PCR, while LH beta could not be amplified, suggesting a possible physiological role of pituitary CG in this species. In conclusion, we demonstrated for the first time that the LH mRNA without exon10 is the natural wild-type LHR in the Platyrrhini lineage. We propose that this LHR represents a new subclass of receptors that should be named LHR type II. In addition, the high expression of CG beta in the marmoset pituitary suggests a physiological role of CG in the reproductive function of these primates beyond pregnancy.     

Molecular cloning of three nonhuman primate follicle stimulating hormone beta-subunit cDNAs

The follicle stimulating hormone (FSH) beta-subunit cDNAs were cloned and sequenced for an old world primate, the rhesus monkey (Macaca mulatta), and two New World primates, the common marmoset (Callithrix jacchus) and pygmy marmoset (Cebuella pygmaea). The cDNA and predicted amino acid sequences of the rhesus monkey FSH beta-subunit were related most closely to the human FSH beta-subunit (> 96% identity). The common and pygmy marmosets have identical FSH beta-subunit cDNAs, whereas the marmoset FSH beta-subunit diverges from the rhesus and human molecules with less than 93% identity. These results have significance for the implementation of assisted reproductive technologies in the nonhuman primate as well as the evolution of genes encoding reproductive hormones.     

Chorionic gonadotropin has a recent origin within primates and an evolutionary history of selection

Chorionic gonadotropin (CG) is a critical signal in establishing pregnancy in humans and some other primates, but this placentally expressed hormone has not been found in other mammalian orders. The gene for one of its two subunits (CG beta subunit [CGbeta]) arose by duplication from the luteinizing hormone beta subunit gene (LHbeta), present in all mammals tested. In this study, 14 primate and related mammalian species were examined by Southern blotting and DNA sequencing to determine where in mammalian phylogeny the CGbeta gene originated. Bats (order Chiroptera), flying lemur (order Dermoptera), strepsirrhine primates, and tarsiers do not have a CGbeta gene, although they possess one copy of the LHbeta gene. The CGbeta gene first arose in the common ancestor of the anthropoid primates (New World monkeys, Old World monkeys, apes, and humans), after the anthropoids diverged from tarsiers. At least two subsequent duplication events occurred in the catarrhine primates, all of which possess multiple CGbeta copies. The LHbeta-CGbeta family of genes has undergone frequent gene conversion among the catarrhines, as well as periods of strong positive selection in the New World monkeys (platyrrhines). In addition, newly generated DNA sequences from the promoter of the CG alpha subunit gene indicate that platyrrhine monkeys use a different mechanism of alpha gene expression control than that found in catarrhines.     

Allelic lineages of the ficolin genes (FCNs) are passed from ancestral to descendant primates

The ficolins recognize carbohydrates and acetylated compounds on microorganisms and dying host cells and are able to activate the lectin pathway of the complement system. In humans, three ficolin genes have been identified: FCN1, FCN2 and FCN3, which encode ficolin-1, ficolin-2 and ficolin-3, respectively. Rodents have only two ficolins designated ficolin-A and ficolin-B that are closely related to human ficolin-1, while the rodent FCN3 orthologue is a pseudogene. Ficolin-2 and ficolin-3 have so far only been observed in humans. Thus, we performed a systematic investigation of the FCN genes in non-human primates. The exons and intron-exon boundaries of the FCN1-3 genes were sequenced in the following primate species: chimpanzee, gorilla, orangutan, rhesus macaque, cynomolgus macaque, baboon and common marmoset. We found that the exon organisation of the FCN genes was very similar between all the non-human primates and the human FCN genes. Several variations in the FCN genes were found in more than one primate specie suggesting that they were carried from one species to another including humans. The amino acid diversity of the ficolins among human and non-human primate species was estimated by calculating the Shannon entropy revealing that all three proteins are generally highly conserved. Ficolin-1 and ficolin-2 showed the highest diversity, whereas ficolin-3 was more conserved. Ficolin-2 and ficolin-3 were present in non-human primate sera with the same characteristic oligomeric structures as seen in human serum. Taken together all the FCN genes show the same characteristics in lower and higher primates. The existence of trans-species polymorphisms suggests that different FCN allelic lineages may be passed from ancestral to descendant species.     

Sequence length polymorphisms within primate amelogenin and amelogenin-like genes: usefulness in sex determination

Sequence length polymorphisms between the amelogenin (AMELX) and the amelogenin-like (AMELY) genes both within and between several mammalian species have been identified and utilized for sex determination, species identification, and to elucidate evolutionary relationships. Sex determination via polymerase chain reaction (PCR) assays of the AMELX and AMELY genes has been successful in greater apes, prosimians, and two species of old world monkeys. To date, no sex determination PCR assay using AMELX and AMELY has been developed for new world monkeys. In this study, we present partial AMELX and AMELY sequences for five old world monkey species (Mandrillus sphinx, Macaca nemestrina, Macaca fuscata, Macaca mulatta, and Macaca fascicularis) along with primer sets that can be used for sex determination of these five species. In addition, we compare the sequences we generated with other primate AMELX and AMELY sequences available on GenBank and discuss sequence length polymorphisms and their usefulness in sex determination within primates. The mandrill and four species of macaque all share two similar deletion regions with each other, the human, and the chimpanzee in the region sequenced. These two deletion regions are 176-181 and 8 nucleotides in length. In analyzing existing primate sequences on GenBank, we also discovered that a separate six-nucleotide polymorphism located approximately 300 nucleotides upstream of the 177 nucleotide polymorphism in sequences of humans and chimps was also present in two species of new world monkeys (Saimiri boliviensis and Saimiri sciureus). We designed primers that incorporate this polymorphism, creating the first AMELX and AMELY PCR primer set that has been used successfully to generate two bands in a new world monkey species.     

Chorionic gonadotrophin and embryo-maternal recognition during the peri-implantation period in primates

Genes for chorionic gonadotrophin (CG) are transcribed by the 16-cell embryo stage in humans, but there is no clear evidence of CG secretion as a bioactive dimer before attachment and trophoblast outgrowth stages of implantation. The studies summarized question the timing of CG expression and secretion, the possible roles of CG for intraembryonic differentiation and at the implantation site, and the recognition of this primate embryo-derived signal in support of the corpus luteum. The data suggest that the implantation window in primates may be broader than in non-primate species, where a closer synchrony between embryonic, tubal and uterine events appears to be necessary for embryonic survival. Some preliminary data concerning an association between peripheral thrombocytopenia, ovarian inhibin secretion and peri-implantation stages of embryo development indicate that an unknown embryonic signal may be secreted before bioactive CG can be detected.     

Homosexual behavior in primates: A review of evidence and theory

Homosexual behavior is defined as genital contact, genital manipulation or both between same-sex individuals. Available data indicate that this behavior is phylogenetically widespread among the anthropoid primates, but totally absent among prosimians. The majority of the 33 species that demonstrate homosexual behavior do so rarefy, but for a substantial number (N =12) it appears to be a more common pattern under free-ranging conditions. I summarize data on homosexual behavior as it relates to form, living condition, age, sex, social organization, and ecological context, and discuss hormonal, demographic, and sociosexual theories for primate homosexual behavior. Among adult primates, the behavior is not the product of abnormal excesses or deficiencies in androgens. Prenatal excesses of androgens may have some effect on the expression of female homosexual behavior, but these effects might vary over the life span, and data are equivocal at present. Demographic processes that result in skewed sex ratios can favor the expression of homosexual behavior in a population, which causes intraspecific variation. I examine several sociosexual explanations, including (a) proceptivity enhancement, (b) receptivity reduction, (c) dominance assertion, (d) practice for heterosexual copulation, (e) tension regulation, (f) reconciliation, and (g) alliance formation. An evolutionary scenario highlights the transformations this behavior underwent during the evolution of the anthropoid primates. I suggest exaptation as a theoretical framework for interpreting homosexual behavior and conclude that future consideration of sexual selection among primates should address homosexual components of this process.     

Molecular evolution of the mammalian ribosomal protein gene, RPS14

Ribosomal protein S14 genes (RPS14) in eukaryotic species from protozoa to primates exhibit dramatically different intron-exon structures yet share homologous polypeptide-coding sequences. To recognize common features of RPS14 gene architectures in closely related mammalian species and to evaluate similarities in their noncoding DNA sequences, we isolated the intron-containing S14 locus from Chinese hamster ovary (CHO) cell DNA by using a PCR strategy and compared it with human RPS14. We found that rodent and primate S14 genes are composed of identical protein-coding exons interrupted by introns at four conserved DNA sites. However, the structures of corresponding CHO and human RPS14 introns differ significantly. Nonetheless, individual intron splice donor, splice acceptor, and upstream flanking motifs have been conserved within mammalian S14 homologues as well as within RPS14 gene fragments PCR amplified from other vertebrate genera (birds and bony fish). Our data indicate that noncoding, intronic DNA sequences within highly conserved, single-copy ribosomal protein genes are useful molecular landmarks for phylogenetic analysis of closely related vertebrate species.      

Evolution of placenta-specific gene expression: comparison of the equine and human gonadotropin alpha-subunit genes.

Primate and equine species are thought to be unique among mammals in synthesizing placental gonadotropin glycoprotein hormones. Human chorionic gonadotropin (CG) and equine pregnant mare's serum gonadotropin (PMSG) are produced in placenta by the specific activation of a glycoprotein hormone alpha-subunit gene and a corresponding beta-subunit gene. The evolutionary mechanisms for the apparently independent acquisition of tissue specificity were investigated by cloning the 5' flanking region of the equine alpha-subunit gene and comparing the DNA elements and trans-acting factors involved in placental expression. We find that though the equine gene is expressed and induced by cAMP, it does not contain the elements known to confer tissue-specific expression to the human gene, the cAMP response element (CRE) and the trophoblast-specific element (TSE), nor does it bind to the trans-acting factors CREB and TSEB. Instead, an additional factor (alpha-ACT) is found which binds to the equine and human, but not the murine, alpha-subunit genes in a region between the positions of the CRE and TSE and confers cAMP responsiveness.     

Primate spermatogenesis: new insights into comparative testicular organisation, spermatogenic efficiency and endocrine control

Owing to the close phylogenetic relationship of Platyrrhini (New World monkeys) and Catarrhini (Old World monkeys) to man, nonhuman primates are often used as models for the study of male reproductive physiology and endocrinology. This review aims at providing new data and insights into comparative primate spermatogenesis, dealing specifically with quantitative aspects of germinal epithelial organisation and germ cell production, and with the roles of gonadotrophic hormones in this process. Typically, the seminiferous epithelium is composed of specific germ cell associations (spermatogenic stages). In rodents, prosimians and most Catarrhini, tubular cross sections contain a single spermatogenic stage whereas in Platyrrhini, great apes and man multi-stage tubules are present. Since Platyrrhini represent a more basal type of primate, this spermatogenic feature must have developed convergently. The primate multi-stage tubular arrangement was previously believed to be associated with low spermatogenic efficiency. However, recent studies using new methodological approaches and comparing primate species from all taxa have revealed that multistage organisation is compatible with highly efficient spermatogenesis. In fact, meta-analysis demonstrated that the efficiency of spermatogenesis in several nonhuman primate species is comparable to that of rodents which are considered as species with highly efficient germ cell production. The duration of the spermatogenic process was not related to organisation or efficiency of spermatogenesis. Sertoli cell work load was species-specific but had no impact on germ cell numbers and on the efficiency of spermatogenesis. The gonadotrophic hormones, luteinizing hormone (LH) and follicle stimulating hormone (FSH) are the primary regulators of primate testicular function. Recent studies revealed that in New World monkeys chorionic gonadotrophin (CG)--the primate pregnancy hormone--regulates testosterone production instead of LH. Receptor studies demonstrated a dual action of the closely related hormones LH and CG in primates. It is hypothesised that following the divergence of the Platyrrhini lineage from Catarrhini, the LH/CG system evolved independently with ancestral functions of the LH/CG system retained in the neotropical taxa. In summary, key spermatogenic features are preserved across all primate taxa whereas male reproductive endocrinology features appear substantially different in the neotropical primates compared to other primate lineages.     

Single-chain, triple-domain gonadotropin analogs with disulfide bond mutations in the alpha-subunit elicit dual follitropin and lutropin activities in vivo

The human glycoprotein hormones chorionic gonadotropin (CG), TSH, LH, and FSH are heterodimers composed of a common alpha-subunit and a hormone-specific beta-subunit. The subunits assemble noncovalently early in the secretory pathway. LH and FSH are synthesized in the same cell (pituitary gonadotrophs), and several of the alpha-subunit sequences required for association with either beta-subunit are different. Nevertheless, no ternary complexes are observed for LH and FSH in vivo, i.e. both beta-subunits assembled with a single alpha-subunit. To address whether the alpha-subunit can interact with more than one beta-subunit simultaneously, we genetically linked the FSHbeta- and CGbeta-subunit genes to the common alpha-subunit, resulting in a single-chain protein that exhibited both activities in vitro. These studies also indicated that the bifunctional triple-domain variant (FSHbeta-CGbeta-alpha), is secreted as two distinct bioactive populations each corresponding to a single activity, and each bearing the heterodimer-like contacts. Although the data are consistent with the known secretion events of gonadotropins from the pituitary, we could not exclude the possibility whether transient intermediates are generated in vivo in which the alpha-subunit shuttles between the two beta-subunits during early stages of accumulation in the endoplasmic reticulum. Therefore, constructs were engineered that would direct the synthesis of single-chain proteins completely devoid of heterodimer-like interactions but elicit both LH and FSH actions. These triple-domain, single-chain chimeras contain the FSHbeta- and CGbeta-subunits and an alpha-subunit with cystine bond mutations (cys10-60 or cys32-84), which are known to prevent heterodimer formation. Here we show that, despite disrupting the intersubunit interactions between the alpha- and both CGbeta- and FSHbeta-subunits, these mutated analogs exhibit both activities in vivo comparable to nonmutated triple-domain single chain. Such responses occurred despite the absence of quaternary contacts due to the disrupted bonds in the alpha-subunit. Thus, gonadotropin heterodimer assembly is critical for intracellular events, e.g. hormone-specific posttranslational modifications, but when heterodimers are present in the circulation, the alpha/beta-contacts are not a prerequisite for receptor recognition.     

Molecular evolution of the primate developmental genes MSX1 and PAX9

In primates, the craniofacial skeleton and the dentition are marked by high levels of interspecific variation. Despite this, there are few comparative species studies conducted at the molecular level to investigate this functional diversity. We have determined nucleotide sequences of MSX1 and PAX9, two developmental genes, in a sample of 27 diverse primate species in order to identify coding or regulatory variation that may be associated with phenotypic diversity. Our analyses have identified four highly conserved noncoding sequences, including one that is conserved across primates and with dogs but not with mice. Although we find that substitution rates vary significantly across MSX1 exons, comparisons of nonsynonymous and synonymous substitution rates (dN/dS) suggest that, as a whole, MSX1 and PAX9 amino acid sequences have been under functional constraint throughout primate evolution. Compared to all other primates in our sample, our analysis of exon 1 in MSX1 finds an unusual pattern of amino acid substitution for Tarsius syrichta, a member of a lineage (tarsiers) that has many unique features among primates. For example, tarsiers are the only extant primates without deciduous incisors, and MSX1 is expressed exclusively in the incisor regions during the earliest stages of dental development. Our overall results provide insight into the utility of comparative species analyses of highly conserved developmental genes and their roles in the evolution of complex phenotypes.     

The beta-subunit of human choriogonadotropin interacts with the exodomain of the luteinizing hormone/choriogonadotropin receptor and changes its interaction with the alpha-subunit.

Human CG (hCG) consists of a common alpha-subunit and a hormone-specific beta-subunit. Similarly, its receptor is also composed of two domains, an extracellular N-terminal half (exodomain) and a membrane-associated C-terminal half (endodomain). hCG initially binds the exodomain of the receptor after which the resulting hCG/exodomain complex is thought to interact with the endodomain. This secondary interaction is considered responsible for signal generation. Despite the importance, it is unclear which hormone subunit interacts with the exodomain or the endodomain. As a step to determine the mechanisms of the initial and secondary interactions and signal generation, we investigated the interaction of the hormone-specific beta-subunit in hCG with the receptor's exodomain. A photoactivable hCG derivative consisting of the wild-type alpha-subunit and a photoactivable beta-subunit derivative was prepared and used to label the exodomain. The analysis and immunoprecipitation of photoaffinity labeled exodomain demonstrate that the beta-subunit in hCG makes the direct contact with the exodomain.     

Consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) for the detection of novel viruses in non-human primates

Consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) have proven to be a powerful tool for the identification of novel genes. CODEHOPs are designed from highly-conserved regions of multiply-aligned protein sequences from members of a gene family and are used in PCR amplification to identify distantly-related genes. The CODEHOP approach has been used to identify novel pathogens by targeting amino acid motifs conserved in specific pathogen families. We initiated a program utilizing the CODEHOP approach to develop PCR-based assays targeting a variety of viral families that are pathogens in non-human primates. We have also developed and further improved a computer program and website to facilitate the design of CODEHOP PCR primers. Here, we detail the method for the development of pathogen-specific CODEHOP PCR assays using the papillomavirus family as a target. Papillomaviruses constitute a diverse virus family infecting a wide variety of mammalian species, including humans and non-human primates. We demonstrate that our pan-papillomavirus CODEHOP assay is broadly reactive with all major branches of the virus family and show its utility in identifying a novel non-human primate papillomavirus in cynomolgus macaques.     

The LHbeta gene of several mammals embeds a carboxyl-terminal peptide-like sequence revealing a critical role for mucin oligosaccharides in the evolution of lutropin to chorionic gonadotropin in the animal phyla

The expression of a previously untranslated carboxylterminal sequence is associated with the ancestral lutropin (LH) beta to the beta-subunit gene evolution of choriogonadotropins (CG). The peptide extension (denoted as CTP) is rich in mucin-type O-glycans and confers new hormonal properties on CG relative to the LH. Although the LHbeta gene is conserved among mammals and only a few frameshift mutations account for the extension, it is merely seen in primates and equids. Bioinformatics identified a CTP-like sequence that is encrypted in the LHbeta gene of several mammalian species but not in birds, amphibians, or fish. We then examined whether or not decoding of the cryptic CTP in the bovine LHbeta gene (boCTP) would be sufficient to generate the LHbeta species of a ruminant with properties typical to the CGbeta subunit. The mutated bovine LHbeta-boCTP subunit was expressed and N-glycosylated in transfected Chinese hamster ovary cells. However, unlike human (h) CGbeta CTP, the cryptic boCTP was devoid of mucin O-glycans. This deficiency was further confirmed when the boCTP domain was substituted for the natural CTP in the human CGbeta subunit. Moreover, when expressed in polarized Madin-Darby canine kidney cells, this hCGbeta-boCTP chimera was secreted basolaterally rather than from the apical compartment, which is the route of the wild type hCGbeta subunit, a sorting function attributed to the O-glycans attached to the CTP. This result shows that the cryptic peptide does not orientate CG to the apical face of the placenta, to the maternal circulation as seen in primates. The absence of this function, which distinguishes CG from LH, provides an explanation as to why the LHbeta to CGbeta evolution did not occur in ruminants. We propose that in primates and equids, further natural mutations in the progenitor LHbeta gene resulted in the efficient O-glycosylation of the CTP, thus favoring the retention of an elongated reading frame.     

The glycoprotein alpha-subunit is critical for secretion and stability of the human thyrotropin beta-subunit

TSH is a member of a family of heterodimeric glycoprotein hormones which have a common alpha-subunit but differ in their hormone-specific beta-subunit. To study the posttranslational processing and assembly of human TSH, eukaryotic expression vectors were constructed that contained either the human TSH beta gene only or both the TSH beta and alpha-genes. These vectors were transfected into Chinese hamster ovary cells and stable cell lines synthesizing TSH beta or TSH dimer were isolated. The kinetics of secretion of TSH beta and the rate of assembly of TSH dimer were compared to the known secretion and assembly of human LH and human CG. In the absence of the alpha-subunit, CG beta is secreted efficiently, but TSH and LH beta-subunits are slowly degraded intracellularly (t1/2 approximately equal to 6 h) and less than 10% is secreted into the medium. In the presence of the alpha-subunit CG beta was also secreted efficiently as dimer but only 50% of the LH beta appeared in the medium as LH dimer. However, unlike LH beta, the alpha-subunit efficiently combines with TSH beta since greater than 95% was secreted as TSH dimer. Thus, the determinants for human TSH beta secretion and assembly are unique from the other human glycoprotein hormone beta-subunits.     

Structure,molecular evolution,and gene expression of primate superoxide dismutases

Mn- and Cu,Zn-superoxide dismutase (SOD) cDNAs of eight primate species, Pan troglodytes, Pongo pygmaeus, Hylobates lar, Macaca fuscata, Macaca fascicularis, Macaca mulatta, Cebus apella, and Callithrix jacchus, were cloned. The whole protein-coding sequences were covered, comparing 198 and 153 (or 154) amino acids, for Mn- and Cu,Zn-SODs, respectively. Residues forming metal ligands were completely conserved in the two primate SODs and nucleotide/amino acid substitutions were more frequent in Cu,Zn-SODs than in Mn-SODs. Molecular evolutionary analyses showed Mn-SOD to have evolved at a constant rate and its phylogenetic tree well reflected primate phylogeny. Cu,Zn-SOD was shown to have evolved differently between primate lineages. The significant high ratio of a non-synonymous/synonymous rate was found in the lineage leading to great apes and humans, showing that this lineage underwent positive Darwinian selection. Southern hybridization suggested that the genes for primate Mn- and Cu,Zn-SOD exist as single copies. Northern analysis in various Japanese monkey tissues showed Mn- and Cu,Zn-SOD expression to be high in the liver, kidneys, and adrenal glands.