Tumor cell membrane cathepsin B.
The lysosomal cysteine peptidase cathepsin B was found to be associated with plasma membrane/endosomal fractions of murine B16 amelanotic melanoma cells. Confocal microscopy with three dimensional image analysis indicated that cathepsin B was associated with the external basal cell surface, which would be consistent with its proposed role in degradation of extracellular matrix proteins. We purified and partially characterized cathepsin B from homogenates of murine liver and B16 amelanotic melanoma cells and from lysosomal and membrane/endosomal fractions of the B16 tumor cells. By SDS-PAGE under reducing conditions, the purified cathepsin B from the tumor homogenates was resolved as a single protein band of Mr 31000, corresponding to the single chain form of cathepsin B. In contrast, cathepsin B from liver homogenates was resolved as two bands of Mr 31000 and 24000, corresponding to the single chain and the heavy chain of the double chain form, respectively. The tumor cathepsin B consisted of four isozymes with pIs of 5.64, 5.33, 5.2 and 5.1, whereas the liver cathepsin B consisted of five isozymes with pIs of 5.64, 5.5, 5.45, 5.35 and 5.3. The additional acidic isoforms of cathepsin B in the B16 tumor probably reflect altered glycosylation in tumors. The commonality of isoforms in the B16 plasma membrane/endosomal and lysosomal fractions suggests that retrograde trafficking of cathepsin B from the lysosome to the endosome and its exocytotic release result in the association of cathepsin B with the tumor cell membrane.
Moin
K
K
Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Cao
L
L
Day
N A
NA
Koblinski
J E
JE
Sloane
B F
BF
D000818:Animals; D015153:Blotting, Western; D002401:Cathepsin B / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D011992:Endosomes / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology; D008546:Melanoma, Experimental / Q000201:enzymology* / Q000473:pathology; D051379:Mice
D002401:Cathepsin B