Use of non-radioactive labels for half-life measurement of sex hormone-binding globulin in the rabbit.
ABSTRACT The purpose of this study was to investigate two methods for labeling rabbit sex hormone-binding globulin (rSHBG) with non-radioactive material, biotin (B) and europium (Eu3+), in order to obtain stable labeled SHBG and measure in vivo its metabolism and distribution. The obtained half-life values were compared with [125I]rSHBG half-lives. rSHBG was first isolated by immunoaffinity chromatography using an immobilized monoclonal anti-human SHBG (hSHBG) antibody that cross-reacts with rSHBG. This purified rSHBG was labeled by either biotin-X-N-hydroxysuccinimide ester (rSHBG-B), Eu3(+)-diethylenetriaminepentaacetic dianhydride, or Eu(3+)-isothiocyanatobenzyldiethylenetriamine-tetraacetic acid reagents (rSHBG-Eu3+) or by 125I using Bolton and Hunter reagent ([125I]rSHBG). The labeling procedure preserved the main properties of native SHBG: interaction with the lectine concanavaline A-Sepharose, recognition by anti-hSHBG monoclonal antibody, and, although lower than in native SHBG, the binding affinity for 5 alpha-dihydrotestosterone. These characteristics were the prerequisite for reliable measurement of the metabolism of labeled SHBG. Labeled rSHBG was injected into various rabbits with blood sampling at 2 min and at 1, 2, 4, 8, 12, 24, 48, 72, and 96 h after injection. rSHBG-B or desiaylated rSHBG-B and rSHBG-Eu3+ were captured from serum samples by tubes coated with anti-hSHBG antibody prior to the following detection procedure: biotin was detected by luminometry with the [streptavidin-alkaline phosphatase-dioxetane (AMPPD)] system and europium by time-resolved fluorimetry. [125I]rSHBG was detected by measurement of radioactivity either directly on serum or after fixation on concanavaline A-Sepharose.(ABSTRACT TRUNCATED AT 250 WORDS)
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ABSTRACT: Sex hormone-binding globulin (SHBG) is the specific plasma transport protein for sex steroid hormones in humans. Considerable variation in SHBG plasma concentration exists between individuals, irrespective of gender, body weight, or thyroid status. In the present work, the influence of carbohydrate chains on the half-life of human SHBG (hSHBG) was investigated using a rabbit model. A variant hSHBG, with a point mutation in exon 8 (GAC --> AAC) encoding an amino acid substitution (Asp327Asn), which introduces an additional consensus site for N-glycosylation, has recently been identified. This mutation suppresses a recognition site for the restriction enzyme Bbs-I, allowing the development of a simple restriction-fragments length polymorphism (RFLP) screening procedure. In a population of patients (272 female and 49 male) consulting in our Endocrinology Clinic, 48 patients (41 female and 7 male) were heterozygous for the variant hSHBG allele and 3 (2 female and 1 male) were homozygous. In this population, the total variant allele frequency was 0.083. The hSHBG genotype, as determined by RFLP, corresponded in all cases to the phenotype as determined by the migration profile of hSHBG by Western blot analysis. The influence of such an additional glycosylation site on the biological half-life of variant hSHBG was investigated. SHBG from serum of patients carrying one of the three hSHBG genotypes was purified and labeled with biotin, then injected into rabbits, as we have recently described for rabbit SHBG. Biotinylated hSHBG was captured from rabbit serum samples on tubes coated with an anti-hSHBG antibody and detected by luminometry with the streptavidine-alkaline phosphatase-dioxetane (AMPPD) system. The results showed that the half-life value was significantly higher (P < 0.05) for SHBG purified from homozygous variant serum (t1/2 beta = 51.43 +/- 1.15 and 63.63 +/- 3.92 h, for male and a female subjects SHBG respectively) than for SHBG purified from heterozygous variant serum (t1/2 beta = 40.19 +/- 0.12 h) or wild-type (t1/2 beta = 38.18 +/- 7.22 h). This study demonstrated that an additional carbohydrate chain on hSHBG decreases the clearance rate of this protein. The low frequency of this variant allele means that further study will be required to determine whether it is associated with higher serum SHBG concentration.Journal of Clinical Endocrinology & Metabolism 01/1998; 83(1):235-40. DOI:10.1210/jcem.83.1.4515 · 6.31 Impact Factor
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ABSTRACT: Human sex hormone-binding globulin (hSHBG) is a plasma glycoprotein that binds sex steroids with high affinity. Variations in hSHBG glycosylation contribute to its electrophoretic microheterogeneity, but the functional significance of different SHBG glycoforms is unknown. Carbohydrates may influence the biological activities and half-lives of glycoproteins and we have examined how oligosaccharides at specific sites influence the plasma clearance of hSHBG in vivo. To accomplish this, fully-glycosylated hSHBG, or hSHBG mutants lacking specific oligosaccharides chains, were expressed in Chinese hamster ovary (CHO) cells and purified by immunoaffinity chromatography. The purified recombinant proteins were then biotinylated to study their plasma half-lives after intravenous injection into rabbits. When compared to hSHBG isolated from serum, recombinant hSHBG migrates with a slightly larger average molecular size during denaturing polyacrylamide gel electrophoresis. This is due to a greater proportion (33-39% vs. 3%) of more highly branched N-linked oligosaccharides on the recombinant proteins. When injected into rabbits, the disappearance of recombinant hSHBG showed two exponential components, as previously shown for natural hSHBG in the same animal model. The mean +/- S.E.M. plasma half-lives of recombinant hSHBG (t 1/2alpha 0.11+/-0.03 h and t 1/2beta 18.94+/-1.65 h) are shorter than previously measured for natural hSHBG (t 1/2alpha 3.43+/-0.72 h and t 1/2beta 38.18+/-7.22 h) and this is likely due to differences in the composition of their N-linked oligosaccharides. An O-linked chain at Thr7 does not influence the plasma clearance of hSHBG in the presence or absence of N-linked carbohydrates at Asn351 and Asn367. However, a 1.5-1.6 fold (p<0.03) increase in plasma half-life of variants lacking both N-glycosylation sites was observed and this is probably due to the fact these variants are not recognized by the asialoglycoprotein receptor-mediated clearance system. Removal of either N-glycosylation consensus site also increased (p<0.0001) the plasma half-life of hSHBG by 2.3 2.4 fold. Thus, the metabolic clearance of hSHBG appears to be determined by the number of N-linked oligosaccharides rather than their location.The Journal of Steroid Biochemistry and Molecular Biology 09/1999; 70(4-6):115-21. DOI:10.1016/S0960-0760(99)00101-6 · 4.05 Impact Factor