Molecular analyses of a human sex hormone-binding globulin variant: evidence for an additional carbohydrate chain.
Genomic DNA was isolated from an individual who is homozygous for a sex hormone-binding globulin (SHBG) variant that resolves into three molecular weight forms of 56K, 52K, and 48K during electrophoresis under denaturing conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). This material was amplified using intron-specific oligonucleotide primers in a polymerase chain reaction to obtain the eight exons encoding SHBG. Sequence analysis of these exons revealed a point mutation encoding an amino acid substitution (Asp --> Asn) at residue 327 in the SHBG polypeptide, and the same mutation was identified in three siblings who also appear to be homozygous for this trait. This mutation introduces an additional consensus site for N-glycosylation at this position, and to confirm its utilization we introduced it into a human SHBG complementary DNA. The mutated complementary DNA was inserted into the pRc/CMV expression vector, and transfected into Chinese hamster ovary (CHO) cells. The product was secreted normally but proportionally less of it (54%) bound to concanavalin A when compared to normal SHBG produced by CHO cells (85%), or SHBG in the serum of either a normal individual or those who produce an electrophoretic variant (98%). Furthermore, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, the SHBG variant produced by CHO cells consisted of a 60K subunit as well as the heavy (52K) and light (48K) subunits associated with normal SHBG produced by CHO cells or in serum. This additional subunit is larger than the variant in serum and probably reflects a greater degree of complexity in the carbohydrate structures added to recombinant SHBG during synthesis in CHO cells. Nevertheless, its steroid-binding affinity was equal to normal SHBG produced by CHO cells or SHBG in serum.
Power
S G
SG
Department of Obstetrics and Gynecology, University of Western Ontario, London, Canada.
Bocchinfuso
W P
WP
Pallesen
M
M
Warmels-Rodenhiser
S
S
Van Baelen
H
H
Hammond
G L
GL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D003208:Concanavalin A / Q000378:metabolism; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D005091:Exons / Q000235:genetics; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012738:Sex Hormone-Binding Globulin / Q000235:genetics* / Q000378:metabolism
D012738:Sex Hormone-Binding Globulin; D003208:Concanavalin A; D004247:DNA