Type IV collagenase and cathepsins L and H : proteinases involved in tumour invasion.
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Date
1992
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Abstract
The collagenolytic proteinases, type IV collagenase and cathepsins Land H, have been
implicated in tumour invasion and metastasis, by virtue of their degradative action on the
extracellular matrix barriers traversed by migrating tumour cells. Type IV collagenase was
isolated from human leucocytes using anti-peptide antibody immunoaffinity
chromatography. The highly specific targeting of both native and denatured forms of human
type IV collagenase by these anti-peptide antibodies holds much promise for
immunolocalisation studies in human tumour tissue. Cathepsin L was purified in both a
free; single-chain form from sheep liver, and as complexes with the endogenous cysteine
proteinase inhibitor, stefin B. These complexes comprised mixtures of the usual tight-binding
non-covalent, inhibitory complexes, and novel, proteolytically active, covalent
cathepsin L/stefin B complexes. The latter form spontaneously in a pH-dependent manner in
vitro from purified, active constituents. The primary structures of these complexing moieties
from sheep liver are reported here for the first time, and showed a high degree of sequence
homology with their human counterparts. Single-chain cathepsin L, both in the free, and
novel, covalently complexed forms, manifested stability and increased activity at neutral pH,
thus suggesting a role in extracellular tissue destruction. This potential involvement in
tumour invasion was strengthened by demonstrating that the single-chain form of the
enzyme, and similar covalent complexes, active under physiological conditions, could be
isolated from liver tissue homogenates of higher primates, baboon (Papio ursinus) and man.
A battery of versatile polyclonal anti-sheep cathepsin L and anti-human cathepsins L
and H peptide antibodies were raised in chickens and rabbits. The chicken egg yolk
antibodies were often of a higher titre than the corresponding rabbit serum antibodies, and
additionally manifested unique immunoinhibitory properties. In the case of the polyclonal
chicken anti-sheep cathepsin L antibodies, this was derived from their ability to target a
peptide located in the active site of cathepsin L. The chicken anti-human cathepsins L and H
peptide antibodies constitute the immunological probes of choice for immunolocalisation and
in vitro tumour invasion studies to elucidate the relative contributions of these collagenolytic
cathepsins to tumour invasion, and could ultimately find application in tumour
immunotherapy.
Description
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1992.
Keywords
Cancer invasiveness., Collagenases., Cysteine proteinases., Proteolytic enzymes., Cathepsin L., Cathepsin H.