Characterization of human type X procollagen and its NC-1 domain expressed as recombinant proteins in HEK293 cells

Frischholz, Svenja, Beier, Frank, Girkontaite, Irute, Wagner, Klaus, Pöschl, Ernst, Turnay, Javier, Mayer, Ulrike ORCID: https://orcid.org/0000-0003-2328-0052 and von der Mark, Klaus (1998) Characterization of human type X procollagen and its NC-1 domain expressed as recombinant proteins in HEK293 cells. Journal of Biological Chemistry, 273 (8). pp. 4547-55. ISSN 0021-9258

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Abstract

Type X collagen is a short-chain, network-forming collagen found in hypertrophic cartilage in the growth zones of long bones, vertebrae, and ribs. To obtain information about the structure and assembly of mammalian type X collagen, we generated recombinant human type collagen X by stable expression of full-length human alpha1(X) cDNA in the human embryonal kidney cell line HEK293 and the fibrosarcoma cell line HT1080. Stable clones were obtained secreting recombinant human type X collagen (hrColX) in amounts of 50 microg/ml with alpha1(X)-chains of apparent molecular mass of 75 kDa. Pepsin digestion converted the native protein to a molecule migrating as one band at 65 kDa, while bands of 55 and 43 kDa were generated by trypsin digestion. Polyclonal antibodies prepared against purified hrColX reacted specifically with type X collagen in sections of human fetal growth cartilage. Circular dichroism spectra and trypsin/chymotrypsin digestion experiments of hrColX at increasing temperatures indicated triple helical molecules with a reduced melting temperature of 31 degrees C as a result of partial underhydroxylation. Ultrastructural analysis of hrColX by rotary shadowing demonstrated rodlike molecules with a length of 130 nm, assembling into aggregates via the globular noncollagenous (NC)-1 domains as reported for chick type X collagen. NC-1 domains generated by collagenase digestion of hrColX migrated as multimers of apparent mass of 40 kDa on SDS-polyacrylamide gel electrophoresis, even after reduction and heat denaturation, and gave rise to monomers of 18-20 kDa after treatment with trichloroacetic acid. The NC-1 domains prepared by collagenase digestion comigrated with NC-1 domains prepared as recombinant protein in HEK293 cells, both in the multimeric and monomeric form. These studies demonstrate the potential of the pCMVsis expression system to produce recombinant triple helical type X collagens in amounts sufficient for further studies on its structural and functional domains.

Item Type:	Article
Uncontrolled Keywords:	cell line,chymotrypsin,electrophoresis, polyacrylamide gel,humans,hydrolysis,immunohistochemistry,microscopy, electron,molecular weight,pepsin a,procollagen,protein conformation,protein denaturation,recombinant proteins,trypsin,tumor cells, cultured
Faculty \ School:	Faculty of Science > School of Biological Sciences
UEA Research Groups:	Faculty of Science > Research Groups > Cells and Tissues Faculty of Medicine and Health Sciences > Research Groups > Gastroenterology and Gut Biology
Depositing User:	Pure Connector
Date Deposited:	13 Jan 2016 12:01
Last Modified:	19 Apr 2023 23:45
URI:	https://ueaeprints.uea.ac.uk/id/eprint/56285
DOI:	10.1074/jbc.273.8.4547

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