Biomaterial Database

Expression and characterization of a serine protease that preferentially cleaves insulin-like growth factor binding protein-5. 2005

J Hou, and D R Clemmons, and S Smeekens

Axys Pharmaceuticals, La Jolla, California, USA.

Insulin-like growth factor binding proteins (IGFBPs) play important roles in regulating the functions of insulin-like growth factors (IGFs). Because IGFBPs have very high affinity for IGF-I and IGF-II, they can regulate the amount of each growth factor that is able to bind to cell surface receptors, therefore, factors that alter IGFBP affinity have the capacity to regulate IGF actions. Protease activities that are present in cell culture systems and physiologic fluids have been shown to degrade IGFBP-5. Previously, a region of sequence in a serine protease was identified that was homologous with the N-terminal 90 amino acids of members of the IGFBP family and with members of the CCN family of proteins. In a prior study, the protease was expressed in human kidney cultured cells and the cell culture supernatants were shown to cleave IGFBP-5, however, it is unknown whether the purified protease would cleave IGFBP-5 and whether it would also cleave other specific forms of IGFBPs. In this study, we expressed this protease in an insect cell expression system, purified it to homogeneity and tested its capacity to cleave IGFBP-5. The expressed protease preferentially cleaved IGFBP-5, and it had minimal activity toward other forms of IGFBPs. The proteolytic activity of this IGFBPase is inhibited by serine protease inhibitors including PMSF and 3,4-dichloroisocoumarin, as well as by divalent metal ions such as, Zn and Cu. Mutation of the active site serine resulted in a major reduction in IGFBP-5 cleavage. The protease binds to heparin and its ability to degrade IGFBP-5 is blocked in the presence of heparin. Inhibition of the activity of the protease following its secretion by B104 cells resulted in inhibition of IGFBP-5 proteolysis and IGF-I stimulation of protein synthesis. Northern blotting revealed that the transcript was expressed in multiple human tissues, including placenta, uterus, prostate, testis, spinal cord, brain, liver, small intestine, thyroid, and spleen. The highest expression was in uterus and placenta, suggesting a possible role of sex steroids in regulating its expression. Understanding the mechanism of how cleavage of IGFBP-5 by this protease alters its activity will help to further our understanding of the biologic actions of the IGFs.

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D006868	Hydrolysis	The process of cleaving a chemical compound by the addition of a molecule of water.
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012697	Serine Endopeptidases	Any member of the group of ENDOPEPTIDASES containing at the active site a serine residue involved in catalysis.	Serine Endopeptidase,Endopeptidase, Serine,Endopeptidases, Serine
D015152	Blotting, Northern	Detection of RNA that has been electrophoretically separated and immobilized by blotting on nitrocellulose or other type of paper or nylon membrane followed by hybridization with labeled NUCLEIC ACID PROBES.	Northern Blotting,Blot, Northern,Northern Blot,Blots, Northern,Blottings, Northern,Northern Blots,Northern Blottings
D015153	Blotting, Western	Identification of proteins or peptides that have been electrophoretically separated by blot transferring from the electrophoresis gel to strips of nitrocellulose paper, followed by labeling with antibody probes.	Immunoblotting, Western,Western Blotting,Western Immunoblotting,Blot, Western,Immunoblot, Western,Western Blot,Western Immunoblot,Blots, Western,Blottings, Western,Immunoblots, Western,Immunoblottings, Western,Western Blots,Western Blottings,Western Immunoblots,Western Immunoblottings
D017386	Sequence Homology, Amino Acid	The degree of similarity between sequences of amino acids. This information is useful for the analyzing genetic relatedness of proteins and species.	Homologous Sequences, Amino Acid,Amino Acid Sequence Homology,Homologs, Amino Acid Sequence,Homologs, Protein Sequence,Homology, Protein Sequence,Protein Sequence Homologs,Protein Sequence Homology,Sequence Homology, Protein,Homolog, Protein Sequence,Homologies, Protein Sequence,Protein Sequence Homolog,Protein Sequence Homologies,Sequence Homolog, Protein,Sequence Homologies, Protein,Sequence Homologs, Protein
D018076	DNA, Complementary	Single-stranded complementary DNA synthesized from an RNA template by the action of RNA-dependent DNA polymerase. cDNA (i.e., complementary DNA, not circular DNA, not C-DNA) is used in a variety of molecular cloning experiments as well as serving as a specific hybridization probe.	Complementary DNA,cDNA,cDNA Probes,Probes, cDNA