Characterization and charge distribution of the asparagine-linked oligosaccharides on secreted mouse thyrotropin and free alpha-subunits.
Mouse hemipituitaries in vitro secrete TSH, composed of an alpha-beta heterodimer, as well as excess (free) alpha-subunits. By dual metabolic labeling with [35S]sulfate and [3H]mannose, we have characterized oligosaccharides from secreted TSH alpha, TSH beta, and free alpha-subunits released from the apoprotein by enzymatic deglycosylation. Oligosaccharides from each subunit displayed a distinct anion exchange HPLC profile due to a specific pattern of sialylation and sulfation. Six species were obtained from TSH alpha (with two glycosylation sites), including neutral oligosaccharides as well as those with one or two negative charges. For TSH beta (with one glycosylation site) at least eight oligosaccharide species were noted, representing nearly every permutation of sialylation and sulfation; approximately 30% contained three or more negative charges. Analysis of [3H]mannose-labeled oligosaccharides on Concanavalin-A-agarose showed 85% binding for those from TSH alpha, 70% for free alpha, and 50% for those from TSH beta. These data demonstrate that oligosaccharides from secreted TSH beta were more sialylated and sulfated, consistent with a more complex branching pattern, than those from TSH alpha. Oligosaccharides from free alpha-subunit were more sialylated than those from TSH alpha, and the net negative charge was intermediate between those of TSH alpha and TSH beta. Although great microheterogeneity is present even at the single glycosylation site on the beta-subunit of secreted TSH, a pattern of sialylation and sulfation could be discerned. If one assigns probabilities of sialylation [p(N)] and sulfation [p(S)] based on the observed distribution within monoacidic (charge -1) species, the proportion of diacidic (charge -2) oligosaccharides could be predicted for each subunit by [p(N)]2, 2[p(N)] [p(S)], [p(S)]2, corresponding to species containing two sialic acid, one sialic acid and one sulfate, and two sulfate residues, respectively. This suggests that the probability of sialylation or sulfation at a second site on these oligosaccharides is similar to that at the first and that anionic oligosaccharides in secreted TSH and free alpha are distributed binomially with regard to sialic acid and sulfate residues.
Gesundheit
N
N
Molecular, Cellular, and Nutritional Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Gyves
P W
PW
DeCherney
G S
GS
Stannard
B S
BS
Winston
R L
RL
Weintraub
B D
BD
D000818:Animals; D001216:Asparagine; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D007037:Hypothyroidism / Q000503:physiopathology; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D051379:Mice; D008815:Mice, Inbred Strains; D009844:Oligosaccharides / Q000032:analysis* / Q000302:isolation & purification; D010902:Pituitary Gland / Q000378:metabolism*; D013961:Thyroid Gland / Q000528:radiation effects; D013972:Thyrotropin / Q000378:metabolism
D046911:Macromolecular Substances; D009844:Oligosaccharides; D001216:Asparagine; D013972:Thyrotropin