PMID: 1400872

 

    Legend: Sugar

Title : Molecular analyses of a human sex hormone-binding globulin variant : evidence for an additional carbohydrate chain

Abstract :
  1. 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)
  2. This material was amplified using intron-specific oligonucleotide primers in a polymerase chain reaction to obtain the eight exons encoding SHBG
  3. 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
  4. 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
  5. The mutated complementary DNA was inserted into the pRc /CMV expression vector, and transfected into Chinese hamster ovary (CHO) cells
  6. 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%)
  7. 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
  8. 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
  9. Nevertheless, its steroid-binding affinity was equal to normal SHBG produced by CHO cells or SHBG in serum