Elevations in cathepsin B protein content and enzyme activity occur independently of glycosylation during colorectal tumor progression.
Western blots, enzyme assays, protein glycosylation studies, and immunohistochemical staining were used to characterize cathepsin B expression at successive stages of colorectal tumor progression. In normal colon mucosa and premalignant adenomas, cathepsin B expression was predominantly due to mature two-chain protein detected on Western blots as the nonglycosylated 27-kDa form, with overexpression of this protein occurring in only 4 of 18 adenomas. Overexpression increased significantly in Dukes A and B carcinomas (26 of 37 cases), with cathepsin B protein generally detectable in carcinomas as a combination of both 27-kDa nonglycosylated and 28-kDa glycosylated mature two-chain forms. Glycosylated cathepsin B protein in carcinoma extracts was sensitive to PNGase F but resistant to Endo H, indicating a pattern consistent with complex rather than high mannose type glycosylation. When sorted by advancing tumor stage, peak expression of cathepsin B protein occurred in carcinomas involved in local invasion compared with adenomas or metastatic cancers. At all stages, cathepsin B activity correlated significantly with the levels of heavy chain mature cathepsin B protein (r = 0.6682, p < 0.0001) irrespective of glycosylation. Immunohistochemical staining of cathepsin B protein revealed fine diffuse cytoplasmic staining in both adenomas and carcinomas compared with coarse granular cytoplasmic staining (typical of lysosomes) seen in matched normal mucosa. Our results demonstrate several sequential, apparently independent changes in cathepsin B expression during colorectal tumor progression including early changes in subcellular localization, up-regulation of cathepsin B protein and activity in invasive cancers, and altered protein glycosylation detected in malignant tumors at all stages.
Iacobuzio-Donahue
C A
CA
Department of Pathology, Boston, Massachusetts 02118, USA.
Shuja
S
S
Cai
J
J
Peng
P
P
Murnane
M J
MJ
D000236:Adenoma / Q000201:enzymology / Q000473:pathology; D015153:Blotting, Western; D002277:Carcinoma / Q000201:enzymology / Q000473:pathology; D002401:Cathepsin B / Q000378:metabolism*; D015179:Colorectal Neoplasms / Q000201:enzymology* / Q000473:pathology; D018450:Disease Progression; D004789:Enzyme Activation; D006031:Glycosylation; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology; D013347:Subcellular Fractions / Q000201:enzymology
D002401:Cathepsin B