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.

Identification of disulfide bonds among the nine core 2 N-acetylglucosaminyltransferase-M cysteines conserved in the mucin beta6-N-acetylglucosaminyltransferase family.

Bovine core 2 beta1,6-N-acetylglucosaminyltransferase-M (bC2GnT-M) catalyzes the formation of all mucin beta1,6-N-acetylglucosaminides, including core 2, core 4, and blood group I structures. These structures expand the complexity of mucin carbohydrate structure and thus the functional potential of mucins. The four known mucin beta1,6-N-acetylglucosaminyltransferases contain nine conserved cysteines. We determined the disulfide bond assignments of these cysteines in [(35)S]cysteine-labeled bC2GnT-M isolated from the serum-free conditioned medium of Chinese hamster ovary cells stably transfected with a pSecTag plasmid. This plasmid contains bC2GnT-M cDNA devoid of the 5'-sequence coding the cytoplasmic tail and transmembrane domain. The C18 reversed phase high performance liquid chromatographic profile of the tryptic peptides of reduced-alkylated (35)S-labeled C2GnT-M was established using microsequencing. Each cystine pair was identified by rechromatography of the C8 high performance liquid chromatographic radiolabeled tryptic peptides of alkylated bC2GnT-M on C18 column. Among the conserved cysteines in bC2GnT-M, the second (Cys(113)) was a free thiol, whereas the other eight cysteines formed four disulfide bridges, which included the first (Cys(73)) and sixth (Cys(230)), third (Cys(164)) and seventh (Cys(384)), fourth (Cys(185)) and fifth (Cys(212)), and eighth (Cys(393)) and ninth (Cys(425)) cysteine residues. This pattern of disulfide bond formation differs from that of mouse C2GnT-L, which may contribute to the difference in substrate specificity between these two enzymes. Molecular modeling using disulfide bond assignments and the fold recognition/threading method to search the Protein Data Bank found a match with aspartate aminotransferase structure. This structure is different from the two major protein folds proposed for glycosyltransferases.

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.

The Ig-like domain of tapasin influences intermolecular interactions.

Presentation of antigenic peptides to T lymphocytes by MHC class I molecules is regulated by events involving multiple endoplasmic reticulum proteins, including tapasin. By studying the effects of substitutions in the tapasin Ig-like domain, we demonstrated that H-2L(d)/tapasin association can be segregated from reconstitution of folded L(d) surface expression. This finding suggests that peptide acquisition by L(d) is influenced by tapasin functions that are independent of L(d) binding. We also found that the presence of a nine-amino acid region in the Ig-like domain of mouse or human tapasin is required for association with L(d), and certain point substitutions in this sequence abrogate human, but not mouse, tapasin association with L(d). These data are consistent with a higher overall affinity between L(d) and mouse tapasin compared with human tapasin. In addition, we found that other point mutations in the same region of the tapasin Ig-like domain affect MHC class I surface expression and Ag presentation. Finally, we showed that the cysteine residues in the Ig-like domain of tapasin influence tapasin's stability, its interaction with the MHC class I H chain, and its stabilization of TAP. Mutagenesis of these cysteines decreases tapasin's electrophoretic mobility, suggesting that these residues form an intramolecular disulfide bond. Taken together, these results reveal a critical role for the tapasin Ig-like domain in tapasin function.

Efficacy of porcine gonadotropins for repeated stimulation of ovarian activity for oocyte retrieval and in vitro embryo production and cryopreservation in Siberian tigers (Panthera tigris altaica).

A comparison of the amino acid sequences demonstrated that Siberian tiger gonadotropins are more homologous with those of porcine than any other commercially available preparation. The present study measured the efficacy of repeated ovarian stimulation with purified porcine gonadotropins on the follicular, hormonal, and immunogenic responses in Siberian tigers as well as on the ability of oocytes retrieved by laparoscopic follicular aspiration to fertilize and cleave in vitro. Controlled rate and vitrification cryopreservation methods were also compared for their ability to support ongoing cleavage following thawing of presumptive 2- to 4-cell tiger embryos generated in vitro. Vitrification supported continued embryonic cleavage in vitro while controlled rate freezing did not. Stereological microscopy indicated an excellent ovarian response with the recovery of quality cumulus-oocyte complexes that apparently fertilized and cleaved in vitro. However, ultrastructural and physiological examination revealed abnormal and unnatural responses such as the failure of some cumulus-oocyte complexes to reach maturity and progestagen levels to approach normalcy. At the same time, analyses of blood for antibodies failed to detect an immune reaction to these foreign gonadotropins in an assay that tested positive for the chorionic gonadotropin-stimulated domestic cat. Together, these observations suggest that porcine gonadotropins may be effective for the ovarian stimulation of tigers but that some modifications to administration protocols are needed to produce a more natural response.

Comparison of chorionic gonadotropin expression in human and macaque (Macaca fascicularis) trophoblasts.

We have designed novel DNA primers that allow us to detect the expression of the subunits of chorionic gonadotropin (CG) from a variety of species of the order Primates. Using these primers, reverse transcriptase-polymerase chain reaction (RT-PCR), and standard cloning techniques, we detected the expression of a single gene for the common glycoprotein hormone (GPH) alpha-subunit and at least two genes for the CG beta-subunit in trophoblasts of Macaca fascicularis (cynomolgous macaque (cm)) at gestational day (GD) = 26 (+/- 2d). No cmCG expression was detected at GD = 35-40. When sequences of cmGPH-alpha and cmCG-beta genes were compared to the corresponding genes of other primates, we found that the alpha-subunit of M. fascicularis was highly conserved compared to other primate species. However, cmCG beta-subunits appeared to be less conserved, residing between those of human CG-beta and baboon CG-beta when analyzed phylogenetically. Of particular interest was a three amino acid stretch in one of the expressed cmCG-beta genes that is distinct from all other primates studied. Our findings imply that not only does the expression of multiple CG beta-subunit genes appear to be common to Old World monkeys, but that the presented methodology will greatly facilitate our ability to understand primate evolution.