The Effect of Desialylation on the in Vitro Interaction of Human Chorionic Gonadotropin with Human Thyroid Plasma Membranes*

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Abstract
Previous studies have indicated that crude preparations of hCG (hCG-c) are much more active than purified preparations (hCG-p) in displacing 125I-labeled bovine TSH ([l25I]iodo-bTSH) from receptors in human and bovine thyroid membranes when the two are compared on the basis of their gonadotropic activity. In an attempt to elucidate the nature of the TSH-displacing factor in hCG-c and in view of the past reports indicating that such preparations contain a spectrum of desialylated hCG molecules, we have desialylated both hCG-c and hCG-p by treatment with neuraminidase and have studied their interactions with human thyroid membranes. Exposure of hCG-c to neuraminidase for increasing lengths of time yielded preparations with progressively greater ability to inhibit the binding of [125I]iodo-bTSH to the membranes, and the increase in inhibitory potency was directly related to the quantity of sialic acid released into the medium. On an equal weight basis, desialylated hCG-c was approximately 5 times more active than intact hCG-c. Though hCG-p was initially far less active than hCG-c in the TSH receptor assay, desialylated forms of the two were almost equal in activity on a unit weight basis. l25I-Labeled asialo-hCG-p ([125I]iodoasialo-hCG-p) exhibited significant direct binding to human thyroid membranes (Kd = 3 ×10-7 M). (For the calculation of molar concentrations, it was assumed that purified bTSH and hCG have bioactivities of 40 and 13,450 IU/tng, respectively. The molecular weights of human TSH, bTSH, and human FSH have been assumed to be 28,000. The following values have been employed for the calculation of molecular weights of hCG and its subunits: hCG or asialo-hCG, 37,000; hCGα or asialo-hCGα 15,000; hCG β or asialo-hCGfr 22,000. The molecular weight values employed for the calculation of molar concentrations of insulin and glucagon are 6,000 and 3,550, respectively.) As judged by their ability to inhibit the binding of [125I]iodoasialo-hCG-p to human thyroid membranes and on an equal weight basis, asialo-hCG-p and hCG-c were approximately 20 and 3 times more active than intact hCG-p and hCG-c, respectively. By the same criterion, hCG-c was approximately 6 times as active as hCG-p. Unlabeled bTSH also inhibited the binding of [125I]iodoasialo-hCG-p, and was approximately 4 times as active as asialo-hCG-p, on a molar basis. Neither crude hCG nor asialo-hCG stimulated adenylate cyclase activity in human thyroid membranes. However, when tested in combination with bTSH, asialo-hCG-p (220 μg/ml), like crude hCG (3000 IU/ml), was a potent antagonist of bTSHinduced stimulation of adenylate cyclase. These findings strongly suggest that 1) desialylation greatly enhances the affinity of hCG for binding to sites in human thyroid membranes; 2) bTSH and asialo-hCG bind to receptors in thyroid that are either the same or interact closely with each other so that the binding of one inhibits the binding of the other; and 3) the factor present in hCG-c that renders it more potent than hCG-p in the TSH radioreceptor assay system may be a population of partially or fully desialylated hCG molecules.

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