Differential Sulfation and Sialylation of Secreted Mouse Thyrotropin (TSH) Subunits: Regulation by TSHReleasing Hormone*

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Abstract
To determine whether sulfate and/or sialic acid are present on secreted mouse TSH, thyrotropic tumor minces and hypothyroid pituitaries were incubated with [3H]methionine and [35S]sulfate, or [35S]methionine and [3H]N-acetylmannosamine. The metabolically labeled TSH and free .alpha.-subunits were than analyzed by gel electrophoresis. [3H]N-Acetylmannosamine was a specific precursor (> 80%) for the sialic acid [3H]N-acetylneuraminic acid, as established by HPLC characterization of tritium label released by acid hydrolysis. Each of the three secreted subunits (TSH.alpha., TSH.beta., and free .alpha.) incorporated both sulfate and sialic acid. The incorporation of these labels was confirmed by the release of [35S]sulfate by endoglycoside F and of [3H]N-acetylneuraminic acid by neuraminidase. Differential labeling of newly synthesized secreted TSH subunits was observed. In secreted TSH dimer, TSH.beta. incorporated 1.3 times more [35S]sulfate (P < 0.05) and 2.5 times more [3H]N-acetylmannosamine (P < 0.02) per carbohydrate chain than did TSH.alpha.. Secreted free .alpha.-subunit incorporated more [3H]N-acetylmannosamine, but less [35S]sulfate, then did secreted TSH.alpha.. To investigate the effect of TRH on TSH sulfation and sialylation, thyrotropic tumor minces and hypothyroid pituitaries were incubated with [35S]sulfate or [3H]N-acetylmannosamine, with or without 10-7 M TRH; labeling was the normalized in each case to incorporation of [3H]mannose, a marker of the inner core sugars. TSH secreted in the presence of TRH had a lower sulfate to mannose ratio [28 .+-. (.+-.SE) 4% of control; P < 0.05] and a lower sialic acid to mannose ratio (63 .+-. 8% of control; P < 0.05). TSH.alpha. and TSH.beta. were affected equally. No change was seen in the labeling of non-TSH secretory proteins. Differential glycoprotein sulfation and sialylation may, in part, explain the previously observed variability in isoelectric point, bioactivity, and MCR of TSH in different physiological states and may represent a point of regulation by TRH.