Cousin P, Déchaud H, Grenot C, Lejeune H, Pugeat M
Laboratoire de la Clinique Endocrinologique, Hôpital de l'Antiquaille, Lyon, France.
J Clin Endocrinol Metab. 1998 Jan;83(1):235-40. doi: 10.1210/jcem.83.1.4515.
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.
性激素结合球蛋白（SHBG）是人体内性类固醇激素的特异性血浆转运蛋白。个体之间SHBG血浆浓度存在显著差异，与性别、体重或甲状腺状态无关。在本研究中，使用兔模型研究了糖链对人SHBG（hSHBG）半衰期的影响。最近发现了一种变异型hSHBG，其外显子8（GAC→AAC）发生点突变，导致氨基酸替代（Asp327Asn），引入了一个额外的N-糖基化共有位点。该突变抑制了限制性内切酶Bbs-I的识别位点，从而开发出一种简单的限制性片段长度多态性（RFLP）筛选程序。在我们内分泌诊所就诊的患者群体（272名女性和49名男性）中，48名患者（41名女性和7名男性）为变异型hSHBG等位基因杂合子，3名患者（2名女性和1名男性）为纯合子。在该群体中，变异等位基因的总频率为0.083。通过RFLP确定的hSHBG基因型在所有情况下均与通过蛋白质印迹分析的hSHBG迁移图谱确定的表型一致。研究了这种额外糖基化位点对变异型hSHBG生物学半衰期的影响。如我们最近对兔SHBG所描述的那样，从携带三种hSHBG基因型之一的患者血清中纯化SHBG并用生物素标记，然后注射到兔子体内。用抗hSHBG抗体包被的试管从兔血清样本中捕获生物素化的hSHBG，并使用链霉亲和素-碱性磷酸酶-二氧杂环丁烷（AMPPD）系统通过发光法进行检测。结果表明，从纯合变异血清中纯化的SHBG的半衰期值（男性和女性受试者的SHBG分别为t1/2β = 51.43±1.15和63.63±3.92小时）显著高于从杂合变异血清中纯化的SHBG（t1/2β = 40.19±0.12小时）或野生型（t1/2β = 38.18±7.22小时）（P < 0.05）。本研究表明，hSHBG上额外的糖链会降低该蛋白的清除率。这种变异等位基因的低频率意味着需要进一步研究以确定它是否与更高的血清SHBG浓度相关。
