Title : Comparison of
cathepsin L synthesized by normal and transformed cells at the gene, message, protein, and oligosaccharide levels
Abstract :
- The major excreted protein of transformed mouse fibroblasts ( MEP ) has recently been identified as the lysosomal cysteine protease , cathepsin L
- The synthesis and intracellular trafficking of this protein in mouse fibroblasts are regulated by growth factors and malignant transformation
- To further define the basis for this regulation, a cDNA encoding MEP / cathepsin L was isolated from a mouse liver cDNA library and used to compare cathepsin L of normal and Kirsten sarcoma virus-transformed NIH 3T3 fibroblasts
- Although cathepsin L message levels were elevated 20-fold in the transformed fibroblasts, normal and transformed cells displayed similar cathepsin L genomic DNA digest patterns and gene copy numbers, and cathepsin L mRNA sequences appeared identical by RNase protection analysis
- These findings indicate that (i) cathepsin L is synthesized from the same gene in normal and transformed cells and (ii) cathepsin L polypeptides made by these cells are translated with the same primary sequence
- Cathepsin L polypeptides synthesized by quiescent, growing, and transformed cells displayed similar isoelectric focusing patterns, suggesting similar post-translational modification
- Site-directed mutagenesis of the mouse liver cDNA and expression in COS monkey cells was used to examine the glycosylation of mouse cathepsin L
- The results indicated that only one of the two potential N-linked glycosylation sites (the one at Asn221 ) is glycosylated
- Analysis by ion exchange chromatography on QAE-Sephadex, and affinity chromatography on mannose 6-phosphate receptor-Affi-Gel 10, indicated that the cathepsin L oligosaccharide was phosphorylated similarly in normal and transformed cells
- Although several phosphorylated oligosaccharide species were observed, the major species contained two phosphomonoester moieties and bound efficiently to the receptor
- These findings suggest that cathepsin L made by normal and transformed mouse fibroblasts are identical and substantiate the hypothesis that trafficking of cathepsin L in these cells is regulated by growth-induced changes in the lysosomal protein transport system
Output (sent_index, trigger,
protein,
sugar,
site):
- 7. glycosylation, , cathepsin L, -, -
- 8. glycosylated, , -, -, sites
- 8. glycosylation, , -, -, sites
Output(Part-Of) (sent_index,
protein,
site):
- 4. cathepsin L mRNA, sequences
- 5. cathepsin L, polypeptides
- 6. Cathepsin L, polypeptides
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):