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 then analyzed by gel electrophoresis. [3H]
N-Acetylmannosamine was a specific precursor (greater than 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
endoglycosidase 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 less than 0.05) and 2.5 times more [3H]
N-acetylmannosamine (P less than 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 then 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 less than 0.05] and a lower
sialic acid to
mannose ratio (63 +/- 8% of control; P less than 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.