Expression and Differential Glycosylation of Human Sex Hormone-Binding Globulin by Mammalian Cell Lines
Wayne P. Bocchinfuso*, Susan Warmels-Rodenhiser, and Geoffrey L. Hammondf Departments of Obstetrics and Gynecology, Biochemistry, and Oncology, and Medical Research Council Group in Fetal and Neonatal Health and Development, University of Western Ontario, London, Ontario, Canada N6A 4L6
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 5a-dihydrotestosterone (K^ = 0.6 nrvi) 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 ConA; this can be explained by the presence of triantennary, rather than biantennary, AMinked 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. (Molecular Endocrinology 5: 1723-1729, 1991)
INTRODUCTION
Sex hormone-binding globulin (SHBG) transports testosterone and estradiol in human blood and functions 0888-8809/91 /1723-1729$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
as a homodimer with a single, high affinity steroidbinding site (1, 2). Plasma SHBG is synthesized in the liver (3), and a structurally related molecule, androgenbinding protein (ABP), is produced in the testis by Sertoli cells (4, 5). These proteins are the products of a single gene located on the short arm of human chromosome 17 (6, 7), and are both glycosylated during synthesis. Although information about the carbohydrate structures associated with human ABP is limited, SHBG contains an O-linked oligosaccharide chain at Thr7 and two Nglycosylation sites that are used within the carboxyterminal portion of the molecule (8, 9). Unlike SHBG, which binds almost quantitatively to Concanavalin-A (Con-A), ABP in human testicular homogenates separates into two forms during Con-A chromatography (10), and these are characterized by minor differences in their peptide maps and the sizes of their respective subunits (11). Like plasma SHBG, the Con-A-binding form of human ABP consists of two subunits with mol wt of 53,000 and 48,000, while ABP that does not interact with Con-A consists of two slightly larger subunits (11). There is no SHBG in adult rat blood, but rat testes also contain two forms of ABP that separate in the same way as human ABP during Con-A chromatography (12). It is, therefore, likely that the Con-A-binding form of human ABP does not simply represent a contamination of testicular homogenates by plasma SHBG, and that both forms of ABP are synthesized in the human testis. Furthermore, because human SHBG and ABP share the same primary structure (6), minor differences between these proteins can probably be attributed to variations in their carbohydrate composition. To study the structure and biosynthesis of SHBG gene products in more detail, we have transfected mammalian cells with an expression vector containing a cDNA that encodes for the human SHBG precursor. This has allowed us to examine the biochemical properties of the recombinant products in the culture medium and to assess cell type-specific differences in their glycosylation.
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The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 07:35 For personal use only. No other uses without permission. . All rights reserved.
MOL ENDO-1991 1724
Vol 5 No. 11
RESULTS Northern Analysis When Chinese hamster ovary (CHO) (13) and BW-1 cells (14) were transfected with a cDNA for human SHBG (Fig. 1 A), both cell lines were found to contain a SHBG mRNA of approximately 1.9 kilobases (kb) in size, while wild-type cells do not contain any crosshybridizing mRNA species (Fig. 1B). The abundance of the SHBG mRNA was assessed with respect to the 18S ribosomal RNA content of these extracts, and densitometric analyses indicated that the CHO cells contain approximately 4 times more SHBG mRNA than the BW-1 cells. By contrast, in this experiment, the SHBG concentration in medium taken from the CHO cells was half of that in BW-1 cell medium. Steroid-Binding Properties of Recombinant SHBG The amount of SHBG was measured in culture medium from transfected CHO and BW-1 cells by an immunoradiometric assay (IRMA) (15) and a steroid-binding ca-
zo
pacity assay (16). The levels of SHBG measured by both assays were equivalent, and medium harvested from confluent cultures contained as much as 4 pmol/ ml. When SHBG produced by CHO cells was examined by Scatchard analysis (17) and compared to SHBG in diluted pregnancy serum, their affinities for 5a-dihydrotestosterone (DHT) were essentially identical; the dissociation constant (K
Wayne P. Bocchinfuso*, Susan Warmels-Rodenhiser, and Geoffrey L. Hammondf Departments of Obstetrics and Gynecology, Biochemistry, and Oncology, and Medical Research Council Group in Fetal and Neonatal Health and Development, University of Western Ontario, London, Ontario, Canada N6A 4L6
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 5a-dihydrotestosterone (K^ = 0.6 nrvi) 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 ConA; this can be explained by the presence of triantennary, rather than biantennary, AMinked 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. (Molecular Endocrinology 5: 1723-1729, 1991)
INTRODUCTION
Sex hormone-binding globulin (SHBG) transports testosterone and estradiol in human blood and functions 0888-8809/91 /1723-1729$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
as a homodimer with a single, high affinity steroidbinding site (1, 2). Plasma SHBG is synthesized in the liver (3), and a structurally related molecule, androgenbinding protein (ABP), is produced in the testis by Sertoli cells (4, 5). These proteins are the products of a single gene located on the short arm of human chromosome 17 (6, 7), and are both glycosylated during synthesis. Although information about the carbohydrate structures associated with human ABP is limited, SHBG contains an O-linked oligosaccharide chain at Thr7 and two Nglycosylation sites that are used within the carboxyterminal portion of the molecule (8, 9). Unlike SHBG, which binds almost quantitatively to Concanavalin-A (Con-A), ABP in human testicular homogenates separates into two forms during Con-A chromatography (10), and these are characterized by minor differences in their peptide maps and the sizes of their respective subunits (11). Like plasma SHBG, the Con-A-binding form of human ABP consists of two subunits with mol wt of 53,000 and 48,000, while ABP that does not interact with Con-A consists of two slightly larger subunits (11). There is no SHBG in adult rat blood, but rat testes also contain two forms of ABP that separate in the same way as human ABP during Con-A chromatography (12). It is, therefore, likely that the Con-A-binding form of human ABP does not simply represent a contamination of testicular homogenates by plasma SHBG, and that both forms of ABP are synthesized in the human testis. Furthermore, because human SHBG and ABP share the same primary structure (6), minor differences between these proteins can probably be attributed to variations in their carbohydrate composition. To study the structure and biosynthesis of SHBG gene products in more detail, we have transfected mammalian cells with an expression vector containing a cDNA that encodes for the human SHBG precursor. This has allowed us to examine the biochemical properties of the recombinant products in the culture medium and to assess cell type-specific differences in their glycosylation.
1723
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 07:35 For personal use only. No other uses without permission. . All rights reserved.
MOL ENDO-1991 1724
Vol 5 No. 11
RESULTS Northern Analysis When Chinese hamster ovary (CHO) (13) and BW-1 cells (14) were transfected with a cDNA for human SHBG (Fig. 1 A), both cell lines were found to contain a SHBG mRNA of approximately 1.9 kilobases (kb) in size, while wild-type cells do not contain any crosshybridizing mRNA species (Fig. 1B). The abundance of the SHBG mRNA was assessed with respect to the 18S ribosomal RNA content of these extracts, and densitometric analyses indicated that the CHO cells contain approximately 4 times more SHBG mRNA than the BW-1 cells. By contrast, in this experiment, the SHBG concentration in medium taken from the CHO cells was half of that in BW-1 cell medium. Steroid-Binding Properties of Recombinant SHBG The amount of SHBG was measured in culture medium from transfected CHO and BW-1 cells by an immunoradiometric assay (IRMA) (15) and a steroid-binding ca-
zo
pacity assay (16). The levels of SHBG measured by both assays were equivalent, and medium harvested from confluent cultures contained as much as 4 pmol/ ml. When SHBG produced by CHO cells was examined by Scatchard analysis (17) and compared to SHBG in diluted pregnancy serum, their affinities for 5a-dihydrotestosterone (DHT) were essentially identical; the dissociation constant (K
