Importance of the content and localization of tyrosine residues for thyroxine formation within the N-terminal part of human thyroglobulin.

Thyroxine (T4) is formed by coupling of iodinated tyrosine residues within thyroglobulin (TG). In mature TG, some iodinated tyrosine residues are involved preferentially in T4 formation. In order to investigate the specific role of various tyrosine residues in T4 formation, N-terminal TG fragments with mutated tyrosine residues were constructed. An N-terminal TG fragment 198 amino acids in size and containing seven tyrosine residues at amino acid positions 5, 29, 89, 97, 107, 130 and 192 was expressed in a baculovirus system. Using site-directed mutagenesis, eight mutant TG fragments were constructed in which different tyrosine residues were replaced by phenylalanine. In the first four TG mutants, one single tyrosine residue (5, 89, 97 or 130) was mutated. In the mutant Y(5,89,97,130)F all of these four tyrosine residues were replaced. The sixth mutant Y(29,89,107,130,192)F contained only tyrosine residues 5 and 97 and the seventh (Y(29,89,97,192)F) contained only tyrosine residues 5, 107 and 130. A TG fragment (Y(5,29,89,97,107,130,192)F) in which all tyrosine residues were replaced by phenylalanine was used as a negative control. After in vitro iodination with lactoperoxidase, specific T4 formation was established in the non-mutated wild-type N-terminal TG fragment. In general the T4 formation in the mutant TG constructs decreased when the total number of tyrosine residues in the 198 amino acid fragment decreased, except fragment Y(29,89,97,192) containing three tyrosine residues, two of them being 5 and 130. Although the rate of T4 formation in this mutated N-terminal TG fragment was lower, the ultimate T4 generation was the same as in the wild-type fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of a functional thyroglobulin fragment in a baculovirus system.

The synthesis is described of an N-terminal thyroglobulin (Tg) polypeptide of 27 kDa, which is capable of hormonogenesis, in a baculovirus system. This polypeptide was made using a 657 bp Tg cDNA cloned from human thyroid RNA by a polymerase chain reaction method. The cDNA contained the information for the Tg signal peptide, the N-terminally located site for thyroid hormone formation and, at the 3' end, a sequence coding for six histidine residues. The fragments produced were purified using a nickel-nitrilotriacetic acid column using these six histidine residues. The products were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and showed two glycosylated fragments of 32 and 34 kDa, both of which started with asparagine. Iodination of the fragments with lactoperoxidase in vitro resulted in the formation of thyroxine (T4). The formation rate of T4 in the fragments was about five times lower than that of the mature Tg dimer of 660 kDa, but ten times more rapid than in bovine serum albumin under the same conditions.