Cathepsin L mediates intracellular ileal digestion of gastric intrinsic factor

M. M. Gordon, T. Howard, M. J. Becich, D. H. Alpers

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

Although acidic proteases of lysosomal origin are implicated in the degradation of intrinsic factor (IF) during cobalamin (cbl) transport across enterocytes and proximal renal tubule cell lines, the enzyme(s) involved in this process is not known. Recombinant (baculovirus-produced) rat 125I- labeled IF (125I-rIF), 43 kDa, added in vivo to the lumen of rat ileum was converted intracellularly to peptides of 33 and 26 kDa. In vitro rat 125I- rIF was degraded to peptides of 33 and 31 kDa by addition of cathepsin L; this conversion was fully inhibited by leupeptin. Western blot analysis using antiserum against denatured native rat IF identified additional cathepsin L degradation products in the 17- to 23-kDa range. In vitro the binding of cobalamin partially inhibited cathepsin L degradation of IF. Rat rIF produced from either insect (Sf9) or mammalian (CHO) cells and native rat IF were all degraded by cathepsin L, although the prominence of the various products differed in the recombinant preparations, being 33 and 36 kDa, respectively. Native rat IF was most sensitive to proteolysis, and no degradation products were identified. Rat 125I-rIF was taken up by LLC-PK1 cells, and 125I from degraded IF appeared abundantly on the basolateral side of cell monolayers by 1 h. The intracellular products of rat rlF in LLC-PK1 cells were the same size as those produced in vitro by the action of cathepsin L. Antiserum against a human kidney cDNA cathepsin L fusion protein easily demonstrated the protease in rat intestinal mucosa, as well as in all other tissues tested. These data suggest that cathepsin L is the protease responsible for the leupeptin-sensitive intracellular degradation of IF.

Original languageEnglish
Pages (from-to)G33-G40
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume268
Issue number1 31-1
StatePublished - Jan 1 1995

Fingerprint Dive into the research topics of 'Cathepsin L mediates intracellular ileal digestion of gastric intrinsic factor'. Together they form a unique fingerprint.

Cite this