Purification and characterization of an incompletely glycosylated form of human chorionic gonadotropin from human placenta.
A small form of hCG (SP-hCG) was purified from an acetone powder preparation of human first trimester placenta by repeated gel filtration and ion exchange chromatography on a Q-Sepharose or FPLC Mono Q column. The estimated mol wt (Mr) of the small hCG by gel filtration is 43K compared to 58K for authentic hCG. The pI of SP-hCG is 10.0, suggesting deficiency of sialic acids. SP-hCG dissociates into subunits when treated with 6 M guanidine-HCl or analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two beta-subunits of SP-hCG were found with estimated Mr of 23K and 20K. Both are distinctly smaller than authentic hCG beta. A single alpha-subunit was found, with an estimated Mr of 21 K. The immunoactivity (8,900-10,000 IU/mg) of highly purified SP-hCG was comparable to that of reference hCG (CR119) determined by a RIA method using anti-hCG antibodies. The hCG/LH receptor-binding activity of SP-hCG is equivalent to that of reference hCG (CR119). Its biological activity is lower than that of reference hCG (approximately 30% or more) assayed by the in vitro stimulation of rat Leydig cells to produce testosterone and cAMP. A high dose is required to attain the same level of stimulation as reference hCG. The amino acid composition of SP-hCG is similar to that of reference hCG, whereas its hexsamine content is significantly lower. Its glucosamine content is about half that in reference hCG, while it completely lacks galactosamine. These findings suggest that SP-hCG is deficient in O-linked oligosaccharide chain in the beta-subunit, and that the N-linked oligosaccharide chains of both subunits are shortened. SP-hCG is one of the principal forms of the hormone present in first trimester placenta and may be a key intermediate in the posttranslational biosynthesis of hCG. Although it lacks O-linked sugar chains and shortened N-linked sugar chains, it possesses substantial biological activity. To have full biological activity, the hCG molecule must contain the complete complement of sugar chains.
Wang
H
H
Center for Biomedical Research, Population Council, New York, New York 10021.
Segal
S J
SJ
Koide
S S
SS
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001681:Biological Assay; D006063:Chorionic Gonadotropin / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D018997:Chorionic Gonadotropin, beta Subunit, Human; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D000242:Cyclic AMP / Q000096:biosynthesis; D005260:Female; D015292:Glycoprotein Hormones, alpha Subunit; D006031:Glycosylation; D006595:Hexosamines / Q000032:analysis; D006801:Humans; D007118:Immunoassay; D007526:Isoelectric Point; D007985:Leydig Cells / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010908:Pituitary Hormones, Anterior / Q000302:isolation & purification; D010920:Placenta / Q000032:analysis*; D011247:Pregnancy; D051381:Rats; D011974:Receptors, LH / Q000378:metabolism; D013739:Testosterone / Q000096:biosynthesis
D000596:Amino Acids; D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D015292:Glycoprotein Hormones, alpha Subunit; D006595:Hexosamines; D010446:Peptide Fragments; D010908:Pituitary Hormones, Anterior; D011974:Receptors, LH; D013739:Testosterone; D000242:Cyclic AMP