Human neutrophil collagenase. A distinct gene product with homology to other matrix metalloproteinases.
We have identified and sequenced a cDNA encoding human neutrophil collagenase from a lambda gt11 cDNA library constructed from mRNA extracted from the peripheral leukocytes of a patient with chronic granulocytic leukemia. The library was screened with an oligonucleotide probe constructed from the putative zinc-binding region of fibroblast collagenase. Eleven positive clones were identified, of which the one bearing the largest insert (2.2 kilobases (kb)) was sequenced. From the nucleotide sequence of the 2.2-kb cDNA clone we have deduced a 467-amino acid sequence representing the entire coding sequence of the enzyme. The deduced protein was confirmed as neutrophil collagenase by conformity with the amino-terminal sequence analyses of three tryptic peptides of purified neutrophil collagenase. The cDNA clone hybridizes to a 3.3-kb mRNA present in RNA extracted from human bone marrow but did not hybridize with RNA isolated from U937 cells induced to differentiate with phorbol myristate acetate. Neutrophil collagenase was found to possess 57% identity with the deduced protein sequence for fibroblast collagenase with 72% chemical similarity. Certain regions of the molecule, including the putative zinc-binding region, are highly conserved. When compared with the published sequence for fibroblast collagenase, neutrophil collagenase contains four additional sites for glycosylation. Medium from COS-7 cells transfected with a pcDNA1 eucaryotic expression vector containing cDNA for neutrophil collagenase degraded type I collagen into the three-quarter, one-quarter fragments characteristic of mammalian interstitial collagenase activity. Thus, definitive evidence based on the cDNA sequence confirms the neutrophil collagenase is a distinct gene product and a member of the family of matrix metalloproteinases.
Hasty
K A
KA
Department of Anatomy, University of Tennessee, Memphis 38104.
Pourmotabbed
T F
TF
Goldberg
G I
GI
Thompson
J P
JP
Spinella
D G
DG
Stevens
R M
RM
Mainardi
C L
CL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D015723:Gene Library; D005796:Genes; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000201:enzymology; D008666:Metalloendopeptidases / Q000235:genetics*; D003012:Microbial Collagenase / Q000097:blood / Q000235:genetics*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology*; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D004273:DNA, Neoplasm; D008666:Metalloendopeptidases; D003012:Microbial Collagenase