Mt. Denhartog et al., IMPORTANCE OF THE CONTENT AND LOCALIZATION OF TYROSINE RESIDUES FOR THYROXINE FORMATION WITHIN THE N-TERMINAL PART OF HUMAN THYROGLOBULIN, European journal of endocrinology, 132(5), 1995, pp. 611-617
Thyroxine (T-4) is formed by coupling of iodinated tyrosine residues w
ithin thyroglobulin (TG). In mature TG, some iodinated tyrosine residu
es are involved preferentially in T-4 formation. In order to investiga
te the specific role of various tyrosine residues in T-4 formation, N-
terminal TG fragments with mutated tyrosine residues were constructed.
An N-terminal TG fragment 198 amino acids in size and containing seve
n tyrosine residues at amino acid positions 5, 29, 89, 97, 107, 130 an
d 192 was expressed in a baculovirus system. Using site-directed mutag
enesis, 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 w
ere replaced. The sixth mutant Y(29, 89, 107, 130, 192)F contained onl
y tyrosine residues 5 and 97 and the seventh (Y(29, 89, 97, 192)F) con
tained 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 iodina
tion with lactoperoxidase, specific T-4 formation was established in t
he non-mutated wild-type N-terminal TG fragment. In general the T-4 fo
rmation in the mutant TG constructs decreased when the total number of
tyrosine residues in the 198 amino acid fragment decreased, except fr
agment Y(29, 89, 97, 192) containing three tyrosine residues, two of t
hem being 5 and 130. Although the rate of T-4 formation in this mutate
d N-terminal TG fragment was lower. the ultimate T-4 generation was th
e same as in the wildtype fragment. This indicates that a preferential
involvement of tyrosines 5 and 130 in thyroid hormonogenesis may exis
t.