HUMAN VARIANT SEX HORMONE-BINDING GLOBULIN (SHBG) WITH AN ADDITIONAL CARBOHYDRATE CHAIN HAS A REDUCED CLEARANCE RATE IN RABBIT

Sex hormone-binding globulin (SHBG) is the specific plasma transport p rotein for sex steroid hormones in humans. Considerable variation in S HBG plasma concentration exists between individuals, irrespective of g ender, body weight, or thyroid status. In the present work, the influe nce of carbohydrate chains on the half-life of human SHBG (hSHBG) was investigated using a rabbit model. A variant hSHBG, with a point mutat ion in exon 8 (GAC --> AAC) encoding an amino acid substitution (Asp32 7Asn), which introduces an additional consensus site for N-glycosylati on, has recently been identified. This mutation suppresses a recogniti on site for the restriction enzyme Bbs-I, allowing the development of a simple restriction-fragments length polymorphism (RFLP) screening pr ocedure. In a population of patients (272 female and 49 male) consulti ng in our Endocrinology Clinic, 48 patients (41 female and 7 male) wer e heterozygous for the variant hSHBG allele and 3 (2 female and 1 male ) were homozygous. In this population, the total variant allele freque ncy 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 i nvestigated. SHBG from serum of patients carrying one of the three hSH BG genotypes was purified and labeled with biotin, then injected into rabbits, as we have recently described for rabbit SHBG. Biotinylated h SHBG was captured from rabbit serum samples on tubes coated with an an ti-hSHBG antibody and detected by luminometry with the streptavidine-a lkaline phosphatase-dioxetane (AMPPD) system. The results showed that the half-life Value was significantly higher (P < 0.05) for SHBG purif ied from homozygous variant serum (t 1/2 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 (t 1/2 beta = 40.19 +/- 0.12 h) or wild-type (t 1/2 beta = 38.18 +/- 7.22 h). This study d emonstrated that an additional carbohydrate chain on hSHBG decreases t he clearance rate of this protein. The low frequency of this variant a llele means that further study will be required to determine whether i t is associated with higher serum SHBG concentration.