Purification, localization, and expression of human intestinal alkaline sphingomyelinase

Abstract

Sphingomyelin (SM) metabolism in the gut may have an impact on colon cancer development. In this study, we purified alkaline sphingomyelinase (alk-SMase) from human intestinal content, and studied its location in the mucosa, expression in colon cancer, and function on colon cancer cells. The enzyme was purified by a series of chromatographies. The molecular mass of the enzyme is 60 kDa, optimal pH is 8.5, and isoelectric point is 6.6. Under optimal conditions, 1 mg of the enzyme hydrolyzed 11 mM SM per hour. The properties of the enzyme are similar to those of rat intestinal alk-SMase but not to those of bacterial neutral SMase. Immunogold electronmicroscopy identified the enzyme on the microvillar membrane in endosome-like structures and in the Golgi complexes of human enterocytes. The expression and the activity of the enzyme were decreased in parallel in human colon cancer tissues compared with the adjacent normal tissue. The enzyme inhibited DNA biosynthesis and cell proliferation dose dependently and caused a reduction of SM in HT29 cells. Intestinal alk-SMase is localized in the enterocytes, downregulated in human colon cancer, and may have antiproliferative effects on colon cancer cells. -Duan, R

Conversion of apical plasma membrane sphingomyelin to ceramide attenuates the intoxication of host cells by cholera toxin

“…In the human gut, alkaline SMase (alk-SMase) is the predominant isoform expressed (Nilsson and Duan, 1999). It is a transmembrane ectoenzyme that localizes both to mucosal surfaces and intracellular vesicles in situ and in HT-29 cells (Duan et al, 2003), although the latter possess a deleterious mutation in the alk-SMase gene (Wu et al, 2004). Immunoblot analysis of T84 extracts using antibodies raised against alk-SMase (Cheng et al, 2002) detects a protein band with a molecular mass of~60-65 kDa (Fig.…”

Effects of bile diversion in rats on intestinal sphingomyelinases and ceramidase

“…Our laboratory has recently cloned intestinal Alk-SMase in both human and rats [3,4] and characterized intestinal N-CDase in the purified form [5]. The enzymes are ecto-enzymes that anchor on the brush border surface via a hydrophobic domain at the C-terminal for Alk-SMase and N-terminal for N-CDase [3,6], and can be released in active form into the gut lumen [3,5,7,8]. We previously showed that Alk-SMase activity was significantly decreased in biopsies of human colon cancer and ulcerative colitis [9,10] and have implications for tumorigenesis in the colon and cholesterol absorption in the small intestine, as recently reviewed [11].…”

“…Ström at Astra Zeneca (Stockholm, Sweden) [21]. Anti-human Alk-SMase antibody was developed in AgriSera AB (Vännäs, Sweden) using purified human Alk-SMase as an antigen [8].…”

Generating Ceramide from Sphingomyelin by Alkaline Sphingomyelinase in the Gut Enhances Sphingomyelin-Induced Inhibition of Cholesterol Uptake in Caco-2 Cells

“…It is an ecto enzyme on the apical surface of the enterocytes [9,31] and can be dissociated into the intestinal lumen in an active form by either bile salt [32] or pancreatic trypsin [33].…”