Biomedical subjects

S Warmels-Rodenhiser

Publications and source records attributed to S Warmels-Rodenhiser.

7 recordsLinked to original sources

PubMed1994

Squirrel monkey corticosteroid-binding globulin: primary structure and comparison with the human protein.

Squirrel monkey (Saimiri sciureus) corticosteroid-binding globulin (CBG) is the product of a 1.6-kilobase mRNA in the liver. Analyses of two overlapping cDNAs revealed that the squirrel monkey CBG precursor comprises 406 amino acids, the first 22 residues of which exhibit 91% identity with the human CBG leader sequence. The mature form of squirrel monkey CBG, therefore, very likely comprises 384 amino acids and has a polypeptide mol wt of 42,854. Compared to human CBG, the squirrel monkey protein contains an additional residue (threonine) at position 144, and the two proteins exhibit 86% sequence identity if this is taken into account. Squirrel monkey CBG contains five consensus sites for N-glycosylation, four of which are located in analogous positions in human CBG, and has two cysteine residues in the same relative positions as the cysteines in human CBG. Unlike CBG in most other species, squirrel monkey CBG appears to circulate as a dimer, and its affinity for glucocorticoids is remarkably low. We, therefore, expressed cDNAs for human and squirrel monkey CBGs in Chinese hamster ovary (CHO) cells and compared the physico-chemical properties of the products with those of the corresponding serum proteins. Squirrel monkey CBG is produced by CHO cells as a dimer, and its subunit size heterogeneity is similar to that associated with CBG in serum. In addition, the cortisol-binding affinity of squirrel monkey CBG produced by CHO cells is similar to that of the natural protein and is 5- to 8-fold lower than that of natural or recombinant human CBG. Mutants in which a threonine at position 144 was either added to human CBG or subtracted from squirrel monkey CBG were also expressed in CHO cells. This demonstrated that this additional amino acid in the squirrel monkey CBG sequence may actively contribute to its propensity for spontaneous dimerization, but does not account for its relatively low steroid-binding affinity.

Amino Acid Sequence↗

PubMed1993

Glycosylation of human corticosteroid-binding globulin at aspargine 238 is necessary for steroid binding.

Human corticosteroid binding-globulin (CBG) is a plasma glycoprotein that binds and regulates the biological activity of glucocorticoids and progesterone. Carbohydrates comprise approximately 25% of its molecular mass being represented by bi- and triantennary N-linked oligosaccharides of the N-acetyllactosamine type. To assess the impact of these carbohydrate chains on CBG production and steroid binding, we mutated a human CBG cDNA so that the six consensus sites for N-glycosylation in the CBG polypeptide were eliminated individually and in various combinations. Expression of the mutant cDNAs in Chinese hamster ovary cells showed that all consensus sites may be utilized during the CBG biosynthesis and that the immunochemical properties of the recombinant glycoproteins are similar to those of CBG isolated from human serum. Removal of sugar chains generally led to a reduction in the secretion of recombinant CBG, but complete removal of N-glycosylation sites did not prevent production or secretion of the protein. Our data indicate that an oligosaccharide linked to Asn238 is essential for steroid binding, and we suggest that an interaction between this sugar chain and the polypeptide may be essential for the creation of a high affinity steroid-binding site. In addition, concanavalin A chromatography of mutants containing only one N-glycosylation site at either Asn74 or Asn238 indicated that processing of the oligosaccharides at these positions is site-specific.

Amino Acid Sequence↗

PubMed1992

Structure/function analyses of human sex hormone-binding globulin by site-directed mutagenesis.

Human sex hormone-binding globulin (hSHBG) and rat androgen-binding protein (rABP) exhibit distinct affinities for sex-steroids. We therefore constructed and expressed a hSHBG/rABP hybrid cDNA encoding the N-terminal portion of hSHBG (205 residues) and the C-terminal portion of rABP (168 residues). The resulting chimera displayed similar steroid-binding characteristics as hSHBG and was recognised by a monoclonal antibody (S1B5) for hSHBG. We then created substitutions at Ser-133, His-136 and Met-139. The Asp-133 and Gln-136 mutants bound steroids in the same way as normal hSHBG while the steroid-binding affinity of Trp-139 was reduced. All three mutants cross-reacted similarly in a hSHBG radioimmunoassay, but Gln-136 was recognised poorly by the S1B5 antibody. These data imply that residues involved in steroid-binding are located within the N-terminal half of hSHBG and include Met-139, and that the S1B5 epitope is located in this region.

Amino Acid Sequence↗

PubMed1992

Selective removal of glycosylation sites from sex hormone-binding globulin by site-directed mutagenesis.

Sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein with high affinity for sex steroid hormones. It contains two N-linked carbohydrate chains and one O-linked oligosaccharide per subunit, but their functional significance is not known. Site-directed mutagenesis of a human SHBG cDNA has enabled us to selectively disrupt the known glycosylation sites individually and in various combinations. The mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells, and it was found that the presence of carbohydrates is not an absolute requirement for the secretion of SHBG from these cells, but the absence of both N-linked oligosaccharides reduced the amount of SHBG in the culture medium. In addition, the affinity and specificity of SHBG for steroid ligands was unaffected by the lack of one or more carbohydrate chains. Proportionally greater amounts (26-31%) of the mutants lacking a single N-linked carbohydrate chain failed to interact with Concanavalin-A. (Con-A) compared to normal SHBG produced by CHO cells (15%). Western analysis demonstrated that both consensus sites for N-glycosylation are used and that the typical heavy [mol wt (M(r)), approximately 51,000] and light (M(r), approximately 47,000) subunit size-heterogeneity was maintained regardless of the absence of an O-linked carbohydrate at residue 7. Furthermore, the SHBG mutants containing only one N-linked oligosaccharide comprise only a single subunit with a M(r) of approximately 47,000. This suggests that the heavy subunit contains two N-linked oligosaccharides, while only one of these sites is used on the light subunit. The M(r) of the various SHBG mutants were also examined by gel filtration, and this indicated that they are all produced as homodimers and that carbohydrates are not involved in subunit association.

Animals↗

PubMed1992

Molecular analyses of a human sex hormone-binding globulin variant: evidence for an additional carbohydrate chain.

Genomic DNA was isolated from an individual who is homozygous for a sex hormone-binding globulin (SHBG) variant that resolves into three molecular weight forms of 56K, 52K, and 48K during electrophoresis under denaturing conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). This material was amplified using intron-specific oligonucleotide primers in a polymerase chain reaction to obtain the eight exons encoding SHBG. Sequence analysis of these exons revealed a point mutation encoding an amino acid substitution (Asp --> Asn) at residue 327 in the SHBG polypeptide, and the same mutation was identified in three siblings who also appear to be homozygous for this trait. This mutation introduces an additional consensus site for N-glycosylation at this position, and to confirm its utilization we introduced it into a human SHBG complementary DNA. The mutated complementary DNA was inserted into the pRc/CMV expression vector, and transfected into Chinese hamster ovary (CHO) cells. The product was secreted normally but proportionally less of it (54%) bound to concanavalin A when compared to normal SHBG produced by CHO cells (85%), or SHBG in the serum of either a normal individual or those who produce an electrophoretic variant (98%). Furthermore, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, the SHBG variant produced by CHO cells consisted of a 60K subunit as well as the heavy (52K) and light (48K) subunits associated with normal SHBG produced by CHO cells or in serum. This additional subunit is larger than the variant in serum and probably reflects a greater degree of complexity in the carbohydrate structures added to recombinant SHBG during synthesis in CHO cells. Nevertheless, its steroid-binding affinity was equal to normal SHBG produced by CHO cells or SHBG in serum.

Amino Acid Sequence↗

PubMed1991

Expression and differential glycosylation of human sex hormone-binding globulin by mammalian cell lines.

The human sex hormone-binding globulin (SHBG) gene is responsible for the production of plasma SHBG by the liver and androgen-binding protein in the testis. Cell-specific glycosylation events during synthesis may account for minor differences in the biochemical properties of SHBG and androgen-binding protein, and we have, therefore, expressed a human SHBG cDNA in chinese hamster ovary (CHO) cells and a mouse hepatoma cell line (BW-1), and compared the products to SHBG in serum. The SHBG produced in this way is a homodimer of subunits that exhibit size microheterogeneity similar to SHBG in human serum, and its affinity for 5 alpha-dihydrotestosterone (Kd = 0.6 nM) and other steroids is essentially identical to that of natural SHBG. When medium from transfected CHO and BW-1 cells was subjected to Concanavalin-A (Con-A) chromatography, the relative amounts of SHBG retained by Con-A were 74% and 86%, respectively. In addition, when SHBG produced by CHO cells was separated into two fractions by Con-A chromatography and analyzed by polyacrylamide gel electrophoresis, SHBG that did not interact with Con-A migrated with a slightly larger apparent mol wt than that of SHBG that binds Con-A; this can be explained by the presence of triantennary, rather than biantennary, N-linked oligosaccharide chains. These data also demonstrate that the subunit microheterogeneity associated with plasma SHBG reflects differences in glycosylation during synthesis, which appear to be cell type specific.

Animals↗

PubMed1988

O6-methylguanine-DNA methyltransferase-defective human cell mutant: O6-methylguanine, DNA strand breaks and cytotoxicity.

We have isolated an isogenic O6-methylguanine (O6-MeG)-DNA methyltransferase-defective mutant from a HeLa cell line. This mutant exhibits excess DNA strand breaks and considerable cytotoxicity after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. The increased frequency of strand breaks after MNNG treatment was not abolished by DNA synthesis inhibitors. We propose that the presence of unrepaired O6-MeG lesions leads to excess strand breaks and these, in turn, are mainly responsible for the cytotoxicity.

Cell Survival↗