title: Pancreatic trypsin cleaves intestinal alkaline sphingomyelinase from mucosa and enhances the sphingomyelinase activity.
abstract: Sphingomyelin (SM) hydrolysis in the gut has implications in colonic tumorigenesis and cholesterol absorption. It is triggered by intestinal alkaline sphingomyelinase (Alk-SMase) that is present in the intestinal mucosa and content. The mechanism by which the enzyme is released into the lumen is not clear. We studied whether trypsin can dissociate Alk-SMase from the mucosa and affect its activity. During luminal perfusion of rat intestine, addition of trypsin to the buffer increased Alk-SMase activity in the perfusate output by about threefold. Treating COS-7 cells transfected with Alk-SMase cDNA with trypsin increased the SMase activity in the medium and reduced that in the cell lysate dose dependently. The appearance of Alk-SMase in the perfusate and culture medium was confirmed by Western blot analysis. The effect of trypsin was blocked by trypsin inhibitor, and neither chymotrypsin nor elastase had a similar effect. We also expressed the full length and COOH-terminal truncated Alk-SMase in COS-7 cells and found that the activity of the full-length enzyme is mainly in the cells, whereas that of the truncated form is mainly in the medium. Both forms were active, but only the activity of the full-length Alk-SMase was enhanced by trypsin. By linking a poly-His tag to the constructed cDNA, we found that the first tryptic site Arg440 upstream of the signal anchor was attacked by trypsin. In conclusion, trypsin cleaves the Alk-SMase at the COOH terminal, releases it from mucosa, and meanwhile enhances its activity. The findings indicate a physiological role of trypsin in SM digestion.
authors.affiliation: Gastroenterology Lab, Biomedical Center B11, Lund University, S-221 84 Lund, Sweden.
authors.forename: Jun
authors.initials: J
authors.lastname: Wu
authors.forename: Fuli
authors.initials: F
authors.lastname: Liu
authors.forename: Ake
authors.initials: A
authors.lastname: Nilsson
authors.forename: Rui-Dong
authors.initials: RD
authors.lastname: Duan
mesh_terms: D000468:Alkalies / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D007413:Intestinal Mucosa / Q000201:enzymology*; D007422:Intestines / Q000201:enzymology*; D010179:Pancreas / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014357:Trypsin / Q000008:administration & dosage / Q000494:pharmacology / Q000502:physiology*
chemical_list: D000468:Alkalies; D010446:Peptide Fragments; D013108:Sphingomyelin Phosphodiesterase; D014357:Trypsin