N. Rao Thotakura

Human insulin-like growth factor binding protein-6 is O-glycosylated.

Insulin-like growth factor binding protein-6 (IGFBP-6) is found in serum, cerebrospinal fluid and conditioned media from human fibroblasts. It has a marked preferential binding affinity for IGF-II over IGF-I. The present study demonstrates that IGFBP-6 purified from human cerebrospinal fluid is O-glycosylated but not N-glycosylated. Enzymatic deglycosylation does not alter the high affinity of IGFBP-6 for IGF-II (Ka 4.4 +/- 2.2 x 10(11) M-1) or its preference for IGF-II over IGF-I. The effect of glycosylation of IGFBP-6 on its secretion, in vivo stability or localization remains to be determined.

The role of carbohydrate in human choriogonadotropin (hCG) action. Effects of N-linked carbohydrate chains from hCG and other glycoproteins on hormonal activity☆

Deglycosylation of gonadotropins and thyrotropin results in a major loss of hormonal bioactivity, while not impairing receptor-binding activity. However, a direct role of the glycan moieties in hormonal signal transduction has not been demonstrated. The addition of carbohydrate chains together with the deglycosylated hormone does not restore the hormonal activity. In contrast, glycopeptides were found to inhibit human choriogonadotropin (hCG)-stimulated adenylyl cyclase activity and hCG binding to its receptor. An inhibition of hCG-stimulated adenylyl cyclase activity but not hCG binding to receptor by glycopeptides specifically from hCG, has previously been reported as a lectin-like membrane component has been implicated in hCG action. In the present study we have shown that glycopeptides and oligosaccharides prepared from hCG, transferrin, fetuin, alpha 1-acid glycoprotein and ovalbumin inhibit the binding of hCG to its receptor. The inhibition was also observed with a highly purified preparation of the receptor, thus suggesting a lack of involvement of other lectin-like membrane components as previously proposed. We suggest that a lectin-like interaction with the hormone, if any, involves the receptor itself. Adenylyl cyclase activity stimulated by hCG, isoproterenol or forskolin was inhibited by oligosaccharides, indicating a non-specific interaction. Our results suggest that Asn-linked oligosaccharide chains from various glycoproteins perturb hCG-receptor interactions through a putative carbohydrate binding site on the receptor.

Estrogen synthetase (aromatase). The cytochrome P-450 component of the human placental enzyme is a glycoprotein

Estrogen synthetase (aromatase) is a cytochrome P-450 enzyme system which converts androgens to estrogens. Although this enzyme has been purified and the cDNA-derived amino acid sequence elucidated, very little is known regarding post-translational modifications of this physiologically crucial enzyme. We show here that the cytochrome P-450 component, P-450ES, purified from human term placental microsomes, is a glycoprotein based on the following evidence: its molecular weight is decreased following treatment with endoglycosidase F, concanavalin A-biotin specifically binds to this protein immobilized on nitrocellulose, and its oligosaccharide composition is consistent with a single N-linked fucosylated complex type carbohydrate chain. In a reconstitution system, the aromatase activity using the endoglycosidase F-treated P-450ES was reduced by about 35-40% relative to the native form, regardless of whether androstenedione or testosterone was used as substrate.

Differential effect of inhibitors of oligosaccharide processing on the secretion of thyrotropin from dispersed rodent pituitary cells

We examined the effect of various inhibitors of oligosaccharide processing on the content and secretion of newly synthesized thyroid-stimulating hormone (TSH) from dispersed hypothyroid rodent pituitary cells. 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin, both inhibitors of glucosidases I and II, decreased intracellular TSH (to 60-76% of control) and secreted TSH (to 60-63% of control) after a 1-hour incubation (pulse) with [35S]methionine and an 8-hour incubation (chase) in isotope-free media. In contrast, deoxymannojirimycin and swainsonine, inhibitors of mannosidase I and II, respectively, increased both intracellular TSH (to 267-309% of control) and secreted TSH (to 192% of control) at 8 hours. TSH oligosaccharides synthesized in the presence of these glucosidase and mannosidase inhibitors were largely sensitive to endo-beta-N-acetylglucosaminidase H (endo H), confirming inhibition of processing. Despite differences in oligosaccharide structure, the in vitro bioactivities of these secreted TSH isoforms were nearly identical. These data confirm and extend previous work performed with 1-deoxynojirimycin suggesting that glucosylated high mannose forms of TSH are more susceptible to intracellular degradation. The novel finding that deoxymannojirimycin and swainsonine increase secreted and total TSH above control levels suggests that non-glucosylated high mannose forms as well as hybrid-type oligosaccharides may facilitate secretion and direct TSH away from a natural degradation pathway.

Regulation and Expression of Thyroid Stimulating Hormone

Thyroid stimulating hormone (TSH) is an anterior pituitary hormone that stimulates thyroid hormone production (1). It is composed of two subunits: the alpha subunit, which is in common with the gonadotropins, luteinizing hormone, and follicle stimulating hormone; and the unique beta subunit, which confers biological specificity to the intact hormone and is expressed exclusively in the thyrotropes of the anterior pituitary (2). Previous studies have indicated that the transcription of the TSHβ gene is stimulated by thyrotropin releasing hormone (TRH) (3–6), phorbol esters (5, 7), and the adenylyl cyclase activator, forskolin, or the cAMP analog 8-bromo-cAMP (4–6, 8), and is inhibited by thyroid hormone (9–15).