ABSTRACT
Type XIX collagen is a minor collagen associated with basement membranes in vascular, neuronal, mesenchymal, and epithelial tissues. We demonstrated that the NC1, C-terminal, domain of collagen XIX inhibits the migration capacities of tumor cells and exerts a strong inhibition of tumor growth. Other basement membrane collagens or derived fragments were measured in biological fluids such as blood and urine of patients and appeared to be useful for diagnosis, prognosis, or treatment monitoring. The aim of this study was to develop and validate methods to measure collagen XIX and its fragments in human cell cultures, tissue extracts, and human biological fluids. For that purpose, we developed real-time PCR, Western blot, and competitive enzyme-linked immunosorbent assays. We demonstrated that the methods developed in this paper are specific for collagen XIX. We showed that it is expressed in human cell cultures, tissue extracts, and various biological fluids. These methods may be used in various human tissue extracts and biological fluids such as serum, amniotic fluid, cord blood, and many other fluids. Collagen XIX or its fragments could constitute new biomarkers for human diseases as well as for diagnosis and/or prognosis.
Subject(s)
Body Fluids/chemistry , Collagen/classification , Collagen/metabolism , Epithelial Cells/metabolism , Fibroblasts/metabolism , Tissue Extracts/chemistry , Cell Line , Collagen/chemistry , Enzyme-Linked Immunosorbent Assay , Epithelial Cells/chemistry , Fibroblasts/chemistry , Gene Expression Regulation/physiology , Humans , Osteosarcoma/chemistry , Osteosarcoma/metabolismABSTRACT
Gene product 8 (gp8, 344 amino acids per monomer) of bacteriophage T4 is one of the baseplate structural proteins. We constructed an expression vector of gp8 and developed a method for purification of recombinant protein. CD spectroscopy showed that gp8 is an alpha/beta type structural protein. Its polypeptide chain consists of nearly 40% beta-structure and 15% alpha-helix. These data agree with results of prediction of secondary structure based on the amino acid sequence of the protein. The sedimentation coefficient under standard conditions (S20,w) is 4.6S. Analytical ultracentrifugation results demonstrated that gp8 in solution has two types of oligomers--dimer and tetramer. The tetramer of gp8 may be included in the wedge (1/6 of the baseplate), and the dimer may be an intermediate product of association.
Subject(s)
Bacteriophage T4/genetics , Glycoproteins/chemistry , Glycoproteins/genetics , Viral Proteins/chemistry , Viral Proteins/genetics , Amino Acid Sequence , Bacteriophage T4/chemistry , Base Sequence , Circular Dichroism , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Genes, Viral , Glycoproteins/isolation & purification , Glycoproteins/metabolism , Molecular Sequence Data , Protein Structure, Secondary , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ultracentrifugation , Viral Proteins/isolation & purification , Viral Proteins/metabolismABSTRACT
Bacteriophage T4 fibritin is a triple-stranded, parallel, segmented alpha-helical coiled-coil protein. Earlier we showed that the C-terminal globular domain (foldon) of fibritin is essential for correct trimerization and folding of the protein. We constructed the chimerical fusion protein W31 in which the fibritin foldon sequence is followed by the small globular non-alpha-helical protein gp31 of the T4 phage. We showed that the foldon is capable of trimerization in the absence of the coiled-coil part of fibritin. A deletion mutant of fibritin (NB1) with completely deleted foldon is unable to fold and trimerize correctly. An excess of this mutant protein did not influence the refolding of fibritin in vitro, and the chimerical protein inhibited this process efficiently. Our conclusion is that the trimerization of the foldon is the initial step of fibritin refolding and is followed by the formation of the coiled-coil structure.
