Abstract
Type XII minicollagen chain association was studied using
baculovirus-directed insect cell expression. Since insect cells
contain low endogenous prolyl 4-hydroxylase activity, the mechanism of
the effect of prolyl hydroxylation on trimer formation in this collagen
could be studied directly by adding recombinant baculoviruses directing
the synthesis of prolyl 4-hydroxylase. Prolyl 4-hydroxylase
was shown to be involved in the trimeric assembly process of type
XII collagen through its α subunit, and thus through its
hydroxylase activity.
The transmembrane protein type XIII collagen was also characterized
by means of insect cell expression, for which purpose new antibodies
against its non-collagenous domains NC2 and NC4 were generated,
together with a pan-collagen antibody against collagenous sequences.
Type XIII collagen α chains were found to form disulphide-bonded
homotrimers, and this was enhanced by prolyl 4-hydroxylation. Analysis
of the disulphide-bonding pattern of the eight cysteine residues
of the α1(XIII) chains revealed that some of the cysteines
in the NC1 domain, and possibly the cysteines at the junction of
the COL1 and NC2 domains, are interchain-linked, while the cysteines in
the NC4 domain are intrachain-linked. The three collagenous domains
of type XIII collagen were shown to be in triple-helical conformation
and have different thermal stabilities, i.e. 38±C for the COL1
domain, 49±C for COL2 and 40±C for COL3.
Furthermore, it was shown that type XIII collagen is oriented
in the plasma membrane of insect cells so that its non-collagenous
N-terminus is intracellular and its mostly collagenous C-terminus is
extracellular. Type XIII collagen was also found to be cleaved into
the insect cell culture medium by a furin-like protease.
The expression of various type XIII collagen deletion variants
suggested that chain recognition and the association of type XIII
collagen α chains into trimers occur in the N-terminal
portion of this molecule. An internal in-frame deletion of residues
63-83 immediately adjacent to the transmembrane domain indicated
that this short ectodomain sequence is necessary for the formation of
disulphide-bonded trimers. Since a sequence homologous with these
deleted residues was also found at the same plasmamembrane-adjacent
location in other collagenous transmembrane proteins, this points
to common features in their chain association.
| Identifer | oai:union.ndltd.org:oulo.fi/oai:oulu.fi:isbn951-42-5746-4 |
| Date | 22 August 2000 |
| Creators | Snellman, A. (Anne) |
| Publisher | University of Oulu |
| Source Sets | University of Oulu |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:eu-repo/semantics/publishedVersion |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess, © University of Oulu, 2000 |
| Relation | info:eu-repo/semantics/altIdentifier/pissn/0355-3221, info:eu-repo/semantics/altIdentifier/eissn/1796-2234 |
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