Characterization of phosphate residues on thyroglobulin

Area of Science:

• Biochemistry
• Molecular Biology
• Endocrinology

Background:

• Follicular 19 S thyroglobulin (Tg) is a large glycoprotein crucial for thyroid hormone synthesis.
• Understanding Tg's post-translational modifications, including phosphorylation, is essential for elucidating thyroid function.
• Previous studies have identified glycosylation as a key modification of thyroglobulin.

Purpose of the Study:

• To characterize the nature and location of phosphate residues in thyroglobulin from different species.
• To investigate the relationship between phosphorylation and glycosylation in thyroglobulin.
• To identify specific amino acid residues involved in thyroglobulin phosphorylation.

Main Methods:

• Radiolabeling of thyroglobulin with [32P]phosphate in vitro using rat thyroid hemilobes, FRTL-5 cells, and bovine thyroid slices.
• Analysis of radiolabeled thyroglobulin using sucrose gradients, gel filtration, immunoprecipitation, and gel electrophoresis.
• Enzymatic deglycosylation, peptide mapping (Pronase, tryptic, cyanogen bromide), acid hydrolysis, and 31P nuclear magnetic resonance (NMR) spectroscopy.

Main Results:

• Thyroglobulin contains approximately 10-12 mol of phosphate per mol of protein, which can be radiolabeled.
• Tunicamycin treatment reduced phosphate incorporation by 50%, indicating a role for glycosylation.
• Phosphate residues are associated with both N-linked oligosaccharides (mannose-containing, sensitive to endo-beta-N-acetylglucosaminidase H) and alkali-labile phosphoserine (3-4/mol) and phosphotyrosine (approx. 2/mol) residues.
• Phosphoserine and phosphotyrosine residues are concentrated in specific tryptic (15-kDa) and cyanogen bromide (24-kDa) peptides, largely devoid of carbohydrate.
• 31P NMR confirmed the presence and heterogeneity of phosphate residues.

Conclusions:

• Thyroglobulin possesses both glycosylation-linked and amino acid-linked phosphate modifications.
• Phosphorylation occurs on specific serine and tyrosine residues within distinct peptide regions of thyroglobulin.
• These findings reveal a complex phosphorylation pattern of thyroglobulin, contributing to its structural and functional characteristics.