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Original Studies |
Laboratoire de la Clinique Endocrinologique (P.C., H.L., M.P., C.B., C.B.), and Laboratoire Central de Biochimie (H.D.), Hôpital de l’Antiquaille, 69321 Lyon Cedex 05; INSERM U 329 (P.C., C.G., M.P.), Hôpital Debrousse, 69005 Lyon, France
Address correspondence and reprint requests to: Michel Pugeat, Laboratoire de la Clinique Endocrinologique, Hôpital de l’Antiquaille, 1 rue de l’Antiquaille, 69321 Lyon Cedex, France. E-mail: laboendo{at}cismsun.univ-lyon1.fr
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Abstract |
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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 (t1/2ß = 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 (t1/2ß = 40.19 ± 0.12 h) or wild-type (t1/2ß = 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. .
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The regulation of SHBG liver production is incompletely understood. Numerous studies have shown the effects of hormones (5, 12, 13, 14) and of nutritional status (15, 16, 17) on SHBG synthesis. However, regulating factors are confusing, notably in women with polycystic ovarian syndrome (PCOS), in whom high insulin and androgen levels are often associated with low SHBG concentration (18, 19). Moreover, considerable variations in serum SHBG concentration exist between individuals, irrespective of gender, body weight, thyroid, or androgen status. Genetic polymorphism in the SHBG gene, altering production and/or metabolism, may also contribute to individual SHBG concentration variation. This might be relevant in public health, when interpreting the recent epidemiological studies that have reported increased cardiovascular risk and high incidence of noninsulin-dependent diabetes in patients with low SHBG levels (20, 21).
The effective role of SHBG glycosylation is not clear. Enzymatic deglycosylation (22) and removal of glycosylation sites by site-directed mutagenesis (23) has little or no effect on steroid-binding or dimerization. The presence of carbohydrate chains has, on the other hand, been said to influence SHBG’s biological half-life and metabolism, as has indeed been shown for many other plasma glycoproteins (24, 25, 26, 27).
The product of a variant allele of the SHBG gene has been identified in human serum (28, 29, 30). This allele presents a point mutation in exon 8 encoding for amino acid substitution (Asp327Asn). This mutation introduces a new consensus site for N-glycosylation (Asn-X-Ser or Thr) there, which is utilized in the variant SHBG molecule (31). SHBG encoded by the variant allele is resolved into three species with differing molecular mass (56, 52, and 48 kDa) when analyzed by polyacrylamide gel electrophoresis (PAGE), whereas normal SHBG resolves only into two subunits of 52 and 48 kDa (30).
In the present work, the Western blotting technique was used to discriminate variant allele carriers, and a restriction-fragments length polymorphism (RFLP) procedure was developed to screen variant SHBG in a population of patients referred to our Clinic. The influence of the additional carbohydrate chain on the half-life of SHBG was investigated using a method recently described in our laboratory for rabbit SHBG (32).
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Screening for the electrophoretic variant of SHBG was performed on plasma samples from patients referred to the Clinique Endocrinologique (Pr. J. Tourniaire, Hopital de l’Antiquaille, Lyon, France) with the following diagnosis: hirsutism (n = 98); 21-hydroxylase deficiency (n = 35); infertile but otherwise normal azoospermic or oligospermic men (n = 32); miscellaneous genetic endocrine disorders (n = 131). Samples were also taken from unaffected relatives of patients (n = 29). Since the frequency of the variant allele was low, 325 subjects had to be included to identify just three homozygous variant subjects, two of whom accepted blood sampling (200 mL) for SHBG purification. Informed written consent was obtained from each subject. The study was approved by the local ethics committee.
Animals
Male New Zealand rabbits, weighing 2734 ± 185 g, purchased from Elevage Scientifique des Dombes (Chatillon sur Chalaronne, France), were housed in individual cages and fed with standard commercial rabbit chow and water ad libitum.
Reagents
125I-SHBG-Coatria assay kits were obtained from bioMerieux SA (Marcy l’Etoile, France). Biotin N-hydroxysuccinimide ester (BIOTIN-X-NHS) was obtained from Calbiochem (Meudon, France); alkaline phosphatase-conjugated streptavidine (AVIDx-AP), disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-tricyclo[3.3.1.13,7]decan}4-yL) phenylphosphate (AMPPD), and Green chemiluminescence amplifier (EMERALD) were obtained from Tropix (Bedford, MA). CNBr-activated Sepharose 4B and dNTP solutions were provided by Pharmacia LKB Biotechnology (Uppsala, Sweden). The ECL-detection kit and Hybond ECL nitrocellulose membrane were from Amersham (Les Ulis, France). Taq DNA polymerase was obtained from ATGC Biotechnologie (Noisy le Grand, France). PCR Primers were synthesized by Eurogentec (Seraing, Belgium). Bbs-I enzyme was obtained from New England Biolabs (Beverly, MA). The chemicals were from Merck (Darmstadt, Germany).
SHBG immunoassay
SHBG concentrations were measured with a specific immunoradiometric assay for hSHBG (125I-SHBG-Coatria).
Electrophoresis and blotting
Western immunoblots (WB) were performed as follows: serum was diluted to a final SHBG concentration of 5 nmol/L in phosphate-buffered saline (PBS; 10 mmol/L, pH 7.4, NaCl 8 g/L, KCl 0.2 g/L). Diluted serum (1 mL) was incubated with a monoclonal anti-hSHBG antibody coupled to Sepharose (25 µL), overnight at 4 C. The affinity sepharose gel was then sedimented by centrifuging and was washed twice by PBS (1 mL). The gel pellet was resuspended in a mixture of PBS (50 µL) and PAGE load buffer (25 µL, Tris HCl 0.05 mol/L pH 6.8, containing ß-mercaptoethanol, SDS, glycerol and bromophenol blue) and heated to 100 C for 2 min. The gel was sedimented, and 10 µL of the supernatant containing SHBG was subjected to an SDS-PAGE with 4% and 10% of acrylamide stacking and resolving gels respectively. Proteins were then transferred electrophoretically onto nitrocellulose membrane. After nonspecific sites had been blocked with nonfat milk in Tris buffer saline (TBS; 0.05 mol/L, pH 7.4, NaCl 0.15 mol/L) for 1 hour, the membrane was incubated overnight with a rabbit-derived polyclonal antiserum against hSHBG (kindly provided by G.L. Hammond, London Regional Cancer Centre, London, Ontario, Canada) diluted in TBS (0.01%). Immunoreactive proteins were detected by a horseradish peroxydase-labeled second antibody detection system (ECL-detection kit) according to the manufacturer’s protocol.
Amplification and analysis of SHBG gene exon 8
Bbs-I-RFLP was performed in all subjects presenting variant SHBG with reduced electrophoretic mobility, in order to identify subjects heterozygous (n = 48) and homozygous (n = 3) for mutation in the SHBG gene exon 8. The RFLP procedure was also performed in 38 subjects with wild-type SHBG.
Genomic DNA was extracted from individuals’ white blood cells by phenol-chloroform and kept at 4 C in Tris 10 mmol/L EDTA 0.1 mmol/L until utilization.
The SHBG gene exon 8 was amplified by polymerase chain reaction (PCR) using oligonucleotide primers designed from the published sequence (8), 5'-CTGGATCCGAGCCACCTTAA and 5'-GCCTGGTACATTGCTAGTGC, forwards and reverse respectively. The PCR was performed in a final reaction volume of 100 µL containing genomic DNA (50 ng), primers (100 pmol each), deoxynucleotide triphosphate (25 nmol each), and Taq DNA polymerase (1 U). Reaction mixtures were overlaid with mineral oil and subjected to 35 cycles of amplification (denaturation at 94 C, annealing at 57 C and extension at 72 C, for 1 min per step).
PCR products were digested by Bbs-I restriction enzyme as follows: 5 IU enzyme in 24 µL of enzyme buffer were added to 25 µL of PCR mixture and incubated overnight at 37 C; then, 9 µL of each sample was completed with 1 µL of load buffer (glycerol 50%, EDTA 0.1 mol/L, SDS 1%, Orange G 1 mg/mL) and subjected to 2% agarose gel electrophoresis. The gel was stained with ethidium bromide and fragments were identified under ultraviolet light.
Direct sequencing of SHBG gene exon 8 was performed after PCR amplification, using an Applied Biosystems 373A sequencer and Taq dye terminator kit (Applied Biosystems, Foster City, CA) in three patients, each carrying one of the three different phenotypes.
SHBG purification
SHBG was purified from individual sera (50–80 mL) of four subjects, one male with normal SHBG genotype, one female with a heterozygous genotype, and one male and one female with a homozygous variant SHBG genotype as identified by RFLP analysis. Purification was performed by immunoaffinity chromatography using an immobilized monoclonal anti-hSHBG antibody on CNBr-activated Sepharose 4B, as previously described (33).
Determination of SHBG half-lives
The hSHBG half-lives were determined according to our previously described method (32). Briefly, purified SHBG was biotin-labeled (SHBG-b) and injected into rabbits (three for each SHBG type) in a marginal ear vein. Blood samples (0.5–2 mL) were drawn from the contralateral ear vein at 2, 10, 20, 30, 40, 50 min and at 1, 2, 4, 8, 12, 24, 48, 72, and 96 h. Sera were stored at -20 C until signal measurement.
Tubes coated with an anti-hSHBG monoclonal mouse immunoglobulin were used to immobilize SHBG-b from rabbit serum samples on a solid phase, and immobilized SHBG-b concentrations were measured by luminometry using the streptavidine-alcaline phosphatase-AMPPD system.
Statistical analysis
Frequencies of regular and variant allele were calculated using
the Hardy-Weinberg law, and phenotype distributions were compared by
the 
-square test.
Comparison of serum SHBG levels in hirsute patients was performed by multifactorial analysis of variance (ANOVA) taking account of SHBG genetic polymorphism and obesity (BMI > 23 Kg/m2). P < 0.05 was considered significant. Results are presented as mean ± SD.
SHBG-b levels in rabbit serum samples were plotted against time, and the resulting curves were analysed by regression analysis using the SigmaPlot software (Jandel Scientific, Erkrath, Germany). The signal disappearance best fitted a two-exponential decrease, and the half-life for each component was calculated.
One-way analysis of variance was used to determine the significance of differences between mean half-life values. Individual comparisons between pairs were performed by Fisher’s protected least significant procedure. P < 0.05 was considered significant. Results are presented as mean ± SD.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Western blot analysis of plasma SHBG (Fig. 1
) showed the electrophoretic mobility of
the three different SHBG phenotypes. Wild-type SHBG (W/W) resolved into
two constituents of 52 and 48 kDa, whereas heterozygous (W/v) and
homozygous (v/v) variant SHBGs showed an additional band of 56 kDa,
which was more abundant in the latter phenotype.
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Bbs-I-digested PCR products of hSHBG gene exon 8 (290
bp) resolved into two fragments of 223 and 67 bp in W/W individuals,
into three fragments of 290, 223, and 67 bp in W/v subjects, and were
not digested in v/v subjects (Fig. 2).
Sequences for SHBG gene exon 8 were examined from genomic DNA in three
subjects, each presenting one of the three distinct phenotypes. Direct
sequencing confirmed the presence of the previously described G-to-A
substitution at codon 327 (31) in the two variant allele carriers (W/W
and W/v) (data not shown).
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SHBG phenotypes (WB) and genotypes (RFLP) were in perfect
concordance. Allele frequency and phenotype distribution are given in
Table 1. In our population of patients,
84.3% had a W/W phenotype, 14.7% were W/v, and 3 patients only were
homozygous for the variant SHBG allele. Allele frequencies were 0.917
and 0.083 for the wild-type and the variant respectively.
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In the screened population, hirsute patients represented the largest group. On multifactorial analysis of variance taking account of the genetic polymorphism of SHBG and obesity, there was no significant difference in SHBG concentration between hirsute patients with the wild type (30.8 ± 17.8 nmol/L; n = 84) and with the heterozygous variant (34.9 ± 17.5 nmol/L; n = 14) SHBG. However, SHBG levels were significantly (P = 0.0017) lower in obese than in nonobese hirsute women (22.8 ± 12.1 vs. 41.2 ± 18.3 nmol/L respectively).
Half-life measurement
The disappearance curves of hSHBG from rabbit serum (Fig. 3) showed the presence of two exponential
components. Protein half-lives were deduced from the two distinct
components on each curve with the following equations:
t1/2
= 0.693/ and t1/2ß = 0.693/ß,
where and ß are the exponential coefficients. Half-life values
measured for the various SHBG types are given in Table 2. Results show that t1/2
was similar for all the SHBGs and that t1/2ß was
significantly higher for SHBG purified from v/v subjects. The mean
half-life value for the three experiments was significantly longer
(P < 0.05) for SHBG purified from female than from
male homozygous variant serum.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The enzymatic restriction method developed in the present work allowed
simple and large-scale screening. We found a perfect concordance
between the presence of an additional heavy chain on SHBG Western blot
and the presence of the D327N mutation in exon 8, suggesting a causal
relationship between phenotype and genotype. In our population of 325
individuals, phenotype distribution was identical between men and
women, in line with an expression of bi-allelic SHBG gene polymorphism
and an autosomal codominant transmission mode (29, 30, 35). As
expressed by the low 2 value (0.338), the observed
distribution was in good agreement with the expected distribution.
The hSHBG half-lives values reported here and rabbit SHBG half-life (32) as measured in rabbit were closed, an observation probably to be explained by the close sequence homology of these two SHBG species (9, 36). The present results were also similar to the data of Longcope et al. (37), who measured hSHBG half-life in Rhesus monkey. However, they were shorter than half-life values determined in Rhesus monkey plasma by Namkung et al. for Maccaca nemestria SHBG (38), which has essentially the same structure as human SHBG (39). This discrepancy suggests that human SHBG clears at a faster rate from rabbit than from primate plasma. Our data indicate clearly that the half-life of the hSHBG variant with an additional carbohydrate chain is longer than that of wild-type SHBG in rabbit. Further studies, ideally using a human or primate model, should investigate what molecular and/or cellular mechanisms are involved in the metabolism of SHBG.
The mean half-life value for variant SHBG purified from the homozygous male subject was shorter than that from the female subject. Sex differences in human SHBG glycan microheterogeneity and in rabbit SHBG oligosaccharide types, have been reported (40, 41). There are also indications from other glycoproteins that sugar side-chains affect plasma clearance rates. For example, the metabolic clearance rate of thyroxine-binding globulin (TBG) has been reported to decrease with increasing sialylation, a chemical process that is up-regulated by estrogens (25). This suggests that sex dependent glycosylation could account for subtle change in SHBG metabolism and function in humans, which were not investigated in the present study. More data will be necessary to settle this.
Differences in glycosylation processing is generally believed to influence the metabolism of glycoprotein hormones by targeting various oligosaccharide receptors, particularly the well-known liver asialoglycoprotein receptor (42). In contrast, specific membrane receptors for glycoprotein hormones have little effect on clearance, being confined to relatively small cell populations (24, 26). In this context, the interaction of differently glycosylated hSHBG preparations with the membrane receptor, described by several groups for hSHBG (43, 44, 45), can make an important contribution to further elucidating the metabolism and function of SHBG. The receptor binding domain of hSHBG is confined to amino acid residues 48 to 57 (46), quite far from the carboxyterminus where N-glycosylation sites are located. However, carbohydrate moiety is required for membrane cell binding (47). The spatial molecular folding of SHBG could mean that residues that are distant in the protein sequence are brought nearer. This important issue of human SHBG physiology will be solved when the tridimensional structure of SHBG is elucidated.
In the hirsute patient group, taking into account obesity, we failed to find variant SHBG associated with higher SHBG plasma concentrations. This result suggests that the concentration of SHBG, at least in this population, is mainly influenced by the regulatory factors of SHBG production (17, 18). The influence of glycosylation on SHBG plasma concentration is an important issue that deserves to be investigated in selected homogenous populations of normal or abnormal individuals with control of hormonal and nutritional factors that have been identified as important in the regulation of SHBG production (2).
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Acknowledgments |
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Footnotes |
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Received May 15, 1997.
Revised September 25, 1997.
Accepted October 3, 1997.
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References |
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p13). Cytogenet cell genet. 54:65–67.[Medline]
6 derivatives of
either testosterone or estradiol as unsubstituted photoaffinity
labeling reagents. Biochemistry. 31:7609–7621.[CrossRef][Medline]
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W/W SHBG, • W/v SHBG, 
and 
, v/v
SHBG isolated from a male and a female respectively (each condition is
expressed by a representative curve of three experiments).