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Abstract

Plasma glutamate carboxypeptidase (PGCP) is a metalloproteinase. It is able to hydrolyze
dipeptides into free amino acids. The enzyme is involved in the release of hormone
thyroxin (T4) from Thyroglobulin (Tg), as suggested by thyroid gland studies. Prediction
from the primary structure of human PGCP suggests the presence of five potential Nglycosylation
sites.
In this thesis we present a study of changing the N-glycosylation of PGCP, and how
this modification influences the localization of PGCP and its extracellular role. The
physiological relevance of PGCP was examined by studying the liberation of extracellular
thyroxin.
Using site-directed mutagenesis of PGCP asparagines on sites 61, 179, 353, 356 and
396 were mutated to alanine to prevent glycosylation. A set of nine mutants were
prepared with one to five times mutated glycosylation sites, each fused with green
fluorescent protein. Expression in transfected HEK 293 and FRTL-5 cell lines showed
vesicular localization. Secretion of the mutants was detected using immunological
methods.
Recombinant PGCP was used for protein characterization. Baculovirus expression
system yielded pure and active PGCP (8 mg/l medium). The recombinant protein was
shown to contain a hybrid or complex type of N-glycosylation. The enzyme was able to
cleave the dipeptide Ser-Met, but its activity was inhibited in the presence of EDTA,
DTPA or bestatin. An activation effect of chloride ions was recognized.
Cathepsin C, an exopeptidase, removes dipeptides from the N-terminus of substrates,
and PGCP hydrolyses dipeptides to amino acids. Fischer rat thyroid cells (FRTL-5) were
used to determine the intracellular distribution of the two enzymes by immunological
methods. The enzymes were observed to colocalize with PDI and Golgin-97, markers of
endoplasmic reticulum and Golgi apparatus, respectively, but their presence in lysosomes
was rare, as shown by the marker, LAMP-2.
PGCP and cathepsin C were secreted from FRTL-5 thyroid cells into the mediun.
Secretion of active cathepsin C was stimulated by TSH, insulin and/or somatostatin, as
determined by hydrolysis of the specific substrate, Gly-Phe-4βMNA.
Hybrid or complex type of N-glycosylation was shown, when glycosylation of the
secreted PGCP was analyzed. Deglycosylation of intracellular PGCP led to a small
decrease in molecular mass, indicating the presence of high mannose N-glycosylation.
Further we show that, in FRTL-5 cell line, TSH, insulin, and/or somatostatin induce
up-regulation of N-acetylglucosaminyltransferase 1, the enzyme responsible for the
initiation of biosynthesis of hybrid and complex N-glycosylation of proteins.
Acting together, PGCP and cathepsin C are able to liberate the hormone thyroxin from
thyroglobulin in a linear, time dependent manner. In vitro experiments proved that
cathepsin C removes up to 12 amino acids from the N-terminus of porcine thyroglobulin,
including a dipeptide with thyroxin on position 5. The newly formed N-terminus, Arg-
Pro-, was not hydrolyzed further by cathepsin C. Using reverse phase HPLC, we showed
that products yielded by cathepsin C were further hydrolyzed by PGCP.
When released enzymes were secreted they were able to liberate thyroxin from porcine
thyroglobulin added to the medium. This liberation can be reduced by synthetic inhibitors
of cysteine proteinases and metalloproteinases.
To support data obtained from in vitro studies and cell culture experiments immunelabeled
slices of mouse thyroid gland were performed to show the presence of
investigated enzymes, PGCP and cathepsin C, in epithelial cells of follicles.