Localization of collagenase at the basal plasma membrane of a human pancreatic carcinoma cell line.
We have recently presented biochemical evidence for collagen and gelatin degrading activities associated with plasma membranes of various human cancer cell lines. In this report we describe the localization of interstitial collagenase at the basal plasma membrane of the human pancreatic cancer cell line RWP-I, using immunofluorescence and ultrastructural immunogold labeling techniques. Collagenase was expressed on the extracellular face of the plasma membrane. Furthermore, the immunogold labeling was concentrated on the long, finger-like microvillous projections typically seen on the basal cell surface, while the short, brush-like projections characteristic of the apical cell surface were unlabeled. When the cytoplasmic face of the membrane was made accessible, the number of reactive sites increased markedly, indicating a high concentration of enzyme at the inner surface of the plasma membrane. When plasma membrane fractions of RWP-I cells were prepared by differential centrifugation, high salt washes virtually failed to extract collagenase activity from the membrane, while detergent extraction with n-octyl glucoside, a detergent used in the purification of integral membrane proteins, yielded soluble collagenase activity. When detergent extracted membrane fractions were passed over an anticollagenase immunoaffinity column, collagenase was specifically bound, as demonstrated by the TCA and TCB degradation of type I collagen by the bound material. Gelatinolytic activity did not bind to the column. Furthermore, immunoprecipitation of 125I-labeled detergent extracts of tumor membranes yielded a single Mr 55,000 band consistent with the zymogen form of the connective tissue collagenase. These morphological and biochemical findings suggest that collagenase is a tightly associated component of the basal plasma membrane, where it occupies a strategic location for directional proteolysis during cell migration and invasion.
Moll
U M
UM
Department of Pathology, State University of New York, Stony Brook 11794.
Lane
B
B
Zucker
S
S
Suzuki
K
K
Nagase
H
H
D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods; D003902:Detergents; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D003012:Microbial Collagenase / Q000378:metabolism*; D008871:Microvilli / Q000201:enzymology; D010190:Pancreatic Neoplasms / Q000201:enzymology* / Q000648:ultrastructure; D014407:Tumor Cells, Cultured
D003902:Detergents; D003012:Microbial Collagenase