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Am J Physiol Gastrointest Liver Physiol 287: G967-G973, 2004. First published June 17, 2004; doi:10.1152/ajpgi.00190.2004
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MUCOSAL BIOLOGY

Pancreatic trypsin cleaves intestinal alkaline sphingomyelinase from mucosa and enhances the sphingomyelinase activity

Jun Wu, Fuli Liu, Åke Nilsson, and Rui-Dong Duan

Gastroenterology Lab, Biomedical Center B11, Lund University, S-221 84 Lund, Sweden

Submitted 26 April 2004 ; accepted in final form 9 June 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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.

sphingomyelin; pancreas; digestion; colon cancer

SPHINGOMYELIN (SM) is present in mammalian cell membranes and in the human diet, mainly in egg, milk, and meat (1, 44). Hydrolysis of SM generates multiple lipid messengers such as ceramide, sphingosine, and sphingosine-1-phosphate, which have regulatory effects on cell proliferation, differentiation, and apoptosis (18–20). In the intestinal tract, digestion of SM may have both physiological and pathophysiological implications. First, supplementary SM in the diet may prevent colonic tumorigenesis (7, 28). Second, SM metabolism in the gut may affect absorption of cholesterol, because both in vivo and in vitro experiments showed that the absorption of cholesterol was inhibited by SM (16, 35). Furthermore, SM in the milk was recently found to stimulate intestinal maturation in neonatal rats (31).

An intestinal mucosal enzyme called alkaline sphingomyelinase (Alk-SMase) was discovered more than 30 years ago by Nilsson (32). We recently purified the enzyme from both rat and human intestinal tract and cloned the human form (4, 8, 9). The enzyme is expressed in the intestinal tract of many species and was also found in human bile (12, 34). The enzyme activity is low in the duodenum, increasing in the proximal part of the jejunum, highest in the middle of jejunum, and declining in the ileum and colon (14). We previously showed that the hydrolysis and absorption of dietary SM occurred in the regions of intestine where the Alk-SMase is abundant, indicating an important role of the enzyme in digestion of SM. Immunogold labeling and sequence analysis demonstrated that the enzyme is located at the surface of microvillar membrane as an ectoenzyme (8, 9). Human intestinal Alk-SMase has 458 amino acid residues with a hydrophobic domain at both NH2 and COOH termini. The NH2-terminal domain is a signal peptide and is cleaved, and that at the COOH terminus is predicted to be a signal anchor (8). However, under physiological conditions, high Alk-SMase activity was identified in the intestinal content (14, 32), and the mechanism by which the enzyme dissociates into the lumen has not been well studied.

It is well known that the pancreas secretes multiple enzymes responsible for digestion of dietary proteins, carbohydrates, and lipids. However, a specific enzyme that hydrolyzes dietary SM has not been identified in pancreatic juice (14, 32); the role of pancreas in digestion of SM has therefore not been considered important. Alk-SMase contains several tryptic sites upstream at the COOH-terminal anchor. The present study was designed to investigate whether pancreatic trypsin could dissociate the Alk-SMase from the mucosa and thereby affect the activity of the enzyme.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Sprague-Dawley rats weighing ~200 g were obtained from Möllegård (Ry, Denmark). COS-7 cells were purchased from American Tissue Culture Collection. SM was purified from bovine milk and labeled with [14C-CH3]choline ([14C-SM]) as described previously (40). The specific activity of the labeled SM is 56 µCi/mg. Plasmid pCDNA4/TO/Myc-His B, Lipofectamine 2000, AccuPrime pfX DNA polymerase, anti-His antibody, and all primers used were purchased from Invitrogen (Paisley, UK). GFX DNA gel band purification kit and enhanced chemiluminescence (ECL) Advance Western blotting kit were obtained from Amersham Biosciences (Uppsala, Sweden). Trypsin (15,900 U/mg protein, type IX), {alpha}-chymotrypsin (54 U/mg protein), elastase, trypsin inhibitor, cell culture mediums, and other chemical agents used were purchased from Sigma (Stockholm, Sweden). The anti-human Alk-SMase antibody was developed by AgreSera (Vännäs, Sweden) as described elsewhere (9).

Animal study. The rats were housed in a temperature-controlled room under a 12:12-h light-dark cycle with free access to water. To compare the capacity of Alk-SMase in the intestinal mucosa and content, the rats were anesthetized by muscular injection of a mixture of ketamin and xylazin (1:2). The whole small intestine from pylorus to cecum was removed. The total length of the small intestine was from 100 to 110 cm and varied with individual rat. The whole small intestine was divided into three parts equal in length. The intestinal content in each part was rinsed with 50 ml 0.15 M saline containing 1 mM benzamidine, and the solution was collected. The intestinal mucosa of each intestinal part was scraped, homogenized, and sonicated as described previously (14). The sonicates were centrifuged at 10,000 relative centrifugal force (rcf) for 10 min, and the supernatant was saved. The Alk-SMase activities in the supernatant and in the intestinal content were determined. The protein concentration in the supernatant was determined, and the total activities in both mucosal portion and intestinal contents were calculated.

For perfusion experiment, the rats were anesthetized as above after fasting overnight. An 8-cm-long segment in the middle part of small intestine was cannulated in both ends. The segment was first perfused with Krebs solution containing (in mM) 110 NaCl, 33 NaHCO3, 1.1 MgCl2, 1.29 CaCl2, and 0.5 Na2HPO4, pH7.4, at a rate of 5 ml/min for 20 min to wash out the Alk-SMase in the lumen, and then it was perfused with a reduced rate to 2 ml/min for 10 min. The perfusate outputs of the final 2 min were collected. The perfusion was then switched to the same solution supplemented with 0.2 mg/ml trypsin at the same rate, and the output was collected every min for three fractions, followed by perfusion with the buffer alone for 10 min. All of the outputs collected were centrifuged at 10,000 rcf for 10 min, and the Alk-SMase activity in the supernatant was determined.

SMase assay. Alk-SMase activity was determined by a method described previously (13). Briefly, 5-µl samples were mixed with 95 µl 50 mM Tris·HCl buffer, pH 9.0, containing 0.15 M NaCl, 2 mM EDTA, 6 mM taurocholate, and 0.80 µM [14C]SM (~8,000 dpm) and incubated at 37°C for 30 min. The reaction was terminated by adding 0.4 ml of chloroform/methanol (2:1) followed by centrifugation at 10,000 rcf for 10 s. An aliquot (100 µl) of the upper phase containing the cleaved phosphocholine was analyzed for radioactivity by liquid scintillation.

Transient expression of Alk-SMase in COS-7 cells. Transient expression of Alk-SMase in COS-7 cells was performed as described (8). The cells were cultured in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal calf serum and 2 mM glutamine in either 25-cm2 flasks or in 96-well plates. The Alk-SMase cDNA was cloned into mammalian expression vector pCDNA4/TO/Myc-His at KpnI and NotI sites. At ~80% confluence, the COS-7 cells were transfected with the constructed plasmid in the presence of Lipofectamine 2000. After 48 h culture, the cells were used for the trypsin-cleaving experiments.

The COOH-terminal 441–458 truncated Alk-SMase cDNA with a poly-His linked to Arg 440 was constructed by PCR using sense primer 5'-tcggtaccgaaagcatgagaggcccggccgtcctc-3' and antisense primer 5'-attctagagtcaatggtgatggtgatgatgggatcccctgctgctgggcgggag-3', where the DNA encoding 6x His tag was underlined. The plasmid pcDNA4/TO/Myc-His with full-length SMase cDNA insert (8) was used as a template in a Mastercycler gradient PCR system (Eppendorf). The PCR program was 95°C for 2 min first, followed by 95°C for 15 s, 55°C for 30 s, and 68°C for 100 s for 25 cycles. The PCR products were isolated electrophoretically on 1% agarose gel and purified by GFX DNA purification kit. The constructed plasmid was then transferred into COS-7 cells as described above.

Effect of trypsin and chymotrypsin on dissociation of Alk-SMase from transfected COS-7 cells. The COS-7 cells transfected with full-length Alk-SMase cDNA were cultured in 25-cm2 flasks for 48 h. The cells were washed first with 1640 medium (serum free) twice and then with 0.2 mg/ml trypsin or chymotrypsin for 30 min. At the end of incubation, the cells were scraped and centrifuged at 4,000 rpm for 10 min. The supernatant was saved. The cell pellets were lysed, and the cell-free extracts were prepared as described previously (26). The Alk-SMase activities in both supernatant and the cell-free extracts were determined.

For studying the dose-dependent effect of trypsin, the transfected COS-7 cells were cultured in 96-well plates with different concentrations of trypsin or chymotrypsin ranging from 0 to 0.5 mg/ml for 30 min. At the end of incubation, an aliquot of the medium was taken for Alk-SMase assay. In another experiment, the cells were treated with 0.5 mg/ml trypsin in the presence of 1 mg/ml trypsin inhibitor.

Western blotting. The Western blot analysis of Alk-SMase was performed as described previously (8, 42). For identification of the enzyme in the rat intestinal perfusate after perfusion with trypsin, the output collected was concentrated by ~12-fold by ultrafiltration through a YM 10 membrane with 10-kDa molecular mass cut-off (Amicon, Beverly, MA). Twenty microliters of the sample were subjected to 10% SDS PAGE. The proteins were transferred to nitrocellulose membrane electrophoretically followed by probing with anti-human Alk-SMase (1:1,000). For identification of the COOH-terminal truncated Alk-SMase with a poly-His linker in the cell culture medium, 10 µl of the medium were directly loaded on 10% SDS-PAGE. The proteins were probed with both anti-human Alk-SMase antibody (1:1,000) and anti-His antibody (1:5,000). After being blocked and washed, the membranes were reacted with either anti-rabbit IgG antibody or anti-mouse IgG antibody, conjugated with horseradish peroxidase for 1 h. The Alk-SMase bands were identified by ECL advance reagents, and the remitted light was recorded on Kodak X-ray films.

Enhancement of Alk-SMase activity by trypsin treatment. To investigate the effect of trypsin on Alk-SMase activity, both full-length and the COOH-terminal truncated Alk-SMases were expressed in COS-7 cells. The culture medium was saved, and the cell-free extracts were prepared as described in Effect of trypsin and chymotrypsin on dissociation of Alk-SMase from transfected COS-7 cells. The Alk-SMase activities in both medium and cell lysate were determined. Because we found that the activity of the full-length Alk-SMase expressed was mainly in the cells and that of the COOH-terminal truncated one was mainly in the medium, to study the potential activation of Alk-SMase by trypsin, the lysate of the cells expressing the full-length Alk-SMase and the medium of the cells expressing the truncated one were therefore incubated with different concentrations of trypsin in 50 mM Tris·HCl buffer containing 20 mM CaCl2, pH 8.0, for 30 min. The changes of Alk-SMase activity after incubation with trypsin were determined. The buffer containing no trypsin was taken as control. In all studies, the activity of trypsin was verified using benzoyl-DL-arginine-p-nitroanilide as substrate (17).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Presence of Alk-SMase in the intestinal mucosa and content. The activities of Alk-SMase in the intestinal content and mucosa in the proximal, middle, and distal parts of the small intestine of rats are shown in Fig. 1. In the proximal part of small intestine, the activity was low in both the mucosa and content. In the middle part of small intestine, the activity in the mucosa was ~9.5-fold higher, and that in the content was 6.2-fold higher than those in the proximal part of the small intestine. The total hydrolytic capacity in the intestinal content of this part was about six times higher than that in the mucosa. In the distal part of small intestine, the activity in the mucosa declined, but that in the content maintained at high levels.



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  		Fig. 1. Comparison of alkaline sphingomyelinase (Alk-SMase) activity in rat intestinal content and mucosa. The whole small intestine was removed and cut into proximal, middle, and distal parts in equal length. The intestinal content was collected, and the mucosa was scraped and homogenized as described in MATERIALS AND METHODS. The Alk-SMase activities in both portions were determined, and the total activities were calculated. Results are means ± SE from 6 rats.

 
Dissociation of Alk-SMase after perfusing trypsin in rat intestinal segment. To examine whether trypsin could dissociate the Alk-SMase from the mucosa, a cannulated segment of rat small intestine was perfused with trypsin. As shown in Fig. 2, top, the addition of trypsin increased the output of Alk-SMase activity in the perfusate by ~300%. The dissociation occurred rapidly, the maximal increase was obtained only 1 min after perfusion, and the increase was declined thereafter. The increased activity in the output was accompanied by an increase in Alk-SMase protein in the perfusate as demonstrated by the Western blot analysis (Fig. 2, bottom).



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  		Fig. 2. Release of Alk-SMase by trypsin perfusion in rat intestinal segment. A segment of small intestine was cannulated. The intestinal segment was perfused first with a large amount of buffer, then with the buffer containing 0.2 mg/ml trypsin, and finally with buffer again. The perfusate was collected and analyzed for Alk-SMase activity (top). The perfusates collected before (fractions 1 and 2) and during trypsin perfusion (fractions 3–5) were subjected to Western blot analysis using anti-human Alk-SMase antibody (bottom).

 
Dissociation of Alk-SMase by trypsin in Alk-SMase transfected COS-7 cells. To further confirm that trypsin is able to cleave Alk-SMase from the cell membrane, Alk-SMase was expressed in COS-7 cells followed by treating the cells with trypsin. In the nontreated control cells, the Alk-SMase activity was low in the culture medium (Fig. 3A) and high in the cell lysate (Fig. 3B). However, incubating the cells with trypsin (0.2 mg/ml) increased the Alk-SMase activity in the medium by ~48-fold accompanied by a 75% reduction of the activity in the cells. Chymotrypsin at the same concentration had no effect on Alk-SMase activity in either medium or cell lysate. Pancreatic elastase also failed to dissociate Alk-SMase from the cells (data not shown). Figure 3, bottom, shows that in the medium of the cells treated with trypsin, Alk-SMase was identified by Western blot analysis (lane 1) as a band corresponding to that found in whole cell lysate (lane 2). However, in the medium of cells without trypsin treatment, the enzyme protein was not detectable (lane 3). The cell vitality after trypsin treatment was examined by 0.4% trypan blue. Under the experimental conditions, few cells were positively stained.



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  		Fig. 3. Dissociation of Alk-SMase expressed in COS-7 cells by trypsin and chymotrypsin. COS-7 Cells transiently transfected with Alk-SMase cDNA were incubated with the medium containing trypsin or chymotrypsin. The Alk-SMase activities in medium (A) and cell lysate (B) were determined. The results are means ± SE from 4 experiments. The medium and cell lysate were subjected to Western blot analysis using anti-human SMase antibody (bottom). Lane 1: medium after trypsin treatment; lane 2: lysate of transfected COS-7 cells, and lane 3: medium from control cells.

 
The trypsin-induced dissociation of Alk-SMase from transfected COS-7 cells was dose dependent (Fig. 4). A threefold increase in Alk-SMase in the medium was already obtained by trypsin at a concentration as low as 0.8 µg/ml. The maximal effect of trypsin occurred at a concentration ~100 µg/ml, and 50% of the maximal effect was seen at ~20 µg/ml. The effect of trypsin (0.5 mg/ml) was abolished by trypsin inhibitor (1 mg/ml). The trypsin-released Alk-SMase activity in the medium was decreased from 3.42 to 0.15 nmol·h–1·ml–1 in the presence of trypsin inhibitor (data not shown).



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  		Fig. 4. Dose-dependent effect of trypsin on Alk-SMase release from transfected COS-7 cells. The COS-7 cells transfected with Alk-SMase cDNA were cultured in a 96-well plate overnight for attachment. After the cells were washed, they were cultured in serum-free 1640 medium containing different concentrations of trypsin for 30 min. At the end of incubation, the Alk-SMase in the medium was analyzed. The results are means ± SE from 3 separate experiments.

 
Release of COOH-terminal anchor truncated Alk-SMase in the medium. Apart from expression of the wild-type Alk-SMase in COS-7 cells, we also expressed a modified Alk-SMase with the COOH-terminal anchor (residue 458–441) truncated with a poly-His linked to the truncated end Arg 440. The design is based on the fact that the residues from 458 to 441 are predicted to be a signal anchor and the residue 440 is an Arg, which is the first tryptic site upstream from the transmembrane anchor. As shown in Fig. 5, after expression in COS-7 cells, the activity of the COOH-terminal truncated Alk-SMase was higher in the medium (Fig. 5A) and lower in the cell lysate (Fig. 5B) compared with the cells that expressed the wild-type (full length) Alk-SMase. The results confirmed that Alk-SMase is hooked on the cell membrane via its COOH-terminal anchor, and cleavage of the anchor causes the dissociation of the enzyme.



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  		Fig. 5. Activities of full-length and COOH-terminal truncated Alk-SMase in transfected COS-7 cells. Both full-length Alk-SMase cDNA and that with the COOH-terminal anchor truncated were expressed in COS-7 cells. The cells were scraped and centrifuged. The supernatant was saved, and the cell-free extracts were prepared. The Alk-SMase activities in the medium (A) and cell lysate (B) were determined. Results are means ± SE from 3 separate experiments.

 
Cleavage of Arg 440. Both full-length Alk-SMase in the cell lysate and the truncated form in the medium were treated with trypsin and then analyzed for activity and by Western blot analysis. As shown in Fig. 6, top, the activity of the full-length Alk-SMase, but not the truncated one, was increased by trypsin in a dose-dependent manner; 20 µg/ml trypsin enhanced the activity by ~50%. Western blot analysis showed that after trypsin treatment, the COOH-terminal truncated enzyme with a poly-His linker could only be identified by anti-Alk-SMase antibody but not by anti-His antibody (Fig. 6, bottom), indicating that the poly-His tag linked to the first tryptic site (Arg 440) has been cleaved by trypsin. The Western blot analysis also shows that trypsin treatment did not induce a detectable change of the enzyme mass.



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  		Fig. 6. Enhancement of Alk-SMase activity by trypsin. COS-7 cells were transfected with the full-length and COOH-terminal (residue 441–458) truncated Alk-SMase with a poly-His tag linked to Arg440. The cell lysate of COS-7 cells that expressed the full-length Alk-SMase and the medium of COS-7 cells that expressed the COOH-terminal truncated Alk-SMase were incubated with different concentrations of trypsin at 37°C for 30 min. At the end of incubation, the Alk-SMase activities in the solutions were determined. The results are means ± SE from 3 separate experiments (top). After trypsin treatment, both the control (CTL) and the trypsin (TPS)-treated samples were subjected to Western blot analysis using anti-His antibody and anti-human Alk-SMase antibody (bottom).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Intestinal Alk-SMase is an important enzyme for digestion of dietary SM. The pancreas, the most crucial organ for digestion of nutritional components, has not been considered to have important roles in the digestion of SM due to the lack of a specific SMase in pancreatic juice (14, 32). In this study, we reported two novel functions of pancreatic trypsin: dissociating intestinal Alk-SMase from the intestinal mucosa and meanwhile increasing the enzyme activity. The findings imply a physiological role of pancreatic trypsin in the digestion of dietary SM in the gut.

Intestinal Alk-SMase was discovered more than 30 years ago (32) and was recently purified and cloned (4, 8, 9). Immunogold labeling studies and sequence analysis indicate that the enzyme is a type of ectoenzyme anchoring in the intestinal mucosa via a predicted hydrophobic domain at the COOH terminus. Under physiological conditions, Alk-SMase activity is found in both mucosal membrane and in the intestinal content (14, 32). In this study, we compared the hydrolytic capacity of Alk-SMase in mucosa and content in rats and found that the total activity in the content was about sixfold higher than that in the mucosa. However, when the enzyme was expressed in COS-7 cells, the activity was much higher in the cells than in the culture medium. These findings indicate that certain factors in the intestinal lumen may dissociate the enzyme from the mucosa. Bile salt and sloughing of the mucosal cells have been previously proposed to be involved in the release of Alk-SMase activity into the lumen (10). Our finding in this study that trypsin dissociates the enzyme from the mucosa is the first report of a specific mechanism that dissociates the enzyme from the brush border.

The dissociating effect of trypsin on Alk-SMase was demonstrated in both in vivo experiments on rat intestine and in vitro experiments with transfected COS-7 cells. In both cases, the increases of the enzyme activity and enzyme protein were confirmed by activity assay and Western blot analysis, respectively. The perfusion study showed that the trypsin-induced dissociation of Alk-SMase was rapid, occurring within minutes. The in vitro cell culture study showed that the effect of trypsin was dose dependent; the concentration giving half-maximal effect is 0.8 µM (20 µg/ml). The dissociation of Alk-SMase by trypsin was not caused by a destructive effect of trypsin on membrane proteins, because trypan blue staining was not positive after trypsin treatment. This is expected because in a standard cell culturing, the concentration of trypsin used to subculture the cells is routinely 2.5 mg/ml, which is much higher than the concentrations used in this study. Under physiological conditions, the concentration of trypsin in the rat small intestinal lumen is ~1–10 µM. In humans, the concentration of trypsin in pancreatic juice after CCK stimulation is ~20 µM and the output of pancreatic trypsin into the intestine in response to a meal or CCK is ~15,000–20,000 U/10 min (~1 mg/10 min) (6). Considering the long exposure time of the intestinal mucosa to trypsin, it is reasonable to believe that trypsin is a physiological factor that dissociates Alk-SMase from the mucosa. The dissociating effect of trypsin is specific, because neither chymotrypsin nor elastase had a similar effect, and the effect of trypsin could be blocked completely by trypsin inhibitor.

In the intestinal tract, both endogenous and exogenous SM is present. The endogenous SM is derived from the bile and from the sloughing of the enterocytes. The exogenous SM is mainly from SM-rich dietary products such as milk, egg, and meat (1, 44) and is estimated to be ~250 mg/day for human (33). It is well known that amphipathic phospholipids in the intestinal tract form mixed micelles with bile salts, which facilitate the hydrolysis of the lipids by water-soluble lipolytic enzymes (2, 3, 41). SM, similar to phosphatidylcholine, is amphipathic and will form mixed micelles with bile salts (15, 30). Although Alk-SMase is an ectoenzyme with the active site located outside the mucosa, the dissociation of the enzyme from the mucosa into the lumen could facilitate the exposure of the substrate in the bile salt micelles to the enzyme and increase the efficiency of SM hydrolysis.

Human Alk-SMase is a protein with 458 residues. Analysis of the amino acid sequence found a predicted signal anchor formed by residue 441–457 at its COOH terminus (8). This prediction has been confirmed, because we have shown that the activity of the COOH-terminal anchor truncated enzyme expressed in COS-7 cells was significantly released in the cell culture medium. Upstream of the COOH-terminal anchor, there are several tryptic sites including R440, R432, K430, R424, R393, R375, R368, K364, and K359 for human Alk-SMase (8). We expressed a form of Alk-SMase with a poly-His tag linked to R440, and then treated the enzyme with trypsin followed by Western blot analysis using both anti-Alk-SMase and anti-His antibodies. We found that the trypsin-treated enzyme was only detectable by anti-Alk-SMase, not by anti-His antibody, indicating that the poly-His tag linked to R440 had been cleaved. The results indicate that R440, the first tryptic site located just above the COOH-terminal anchor, is cleaved by trypsin. But our results cannot exclude the possibility that other tryptic sites above R440 can also been cleaved. However, because Western blot analysis of the Alk-SMase treated with trypsin did not show a remarkable reduction in molecular mass, other tryptic sites, if cleaved by trypsin also, are not located far from R440.

In this study, we also found that trypsin not only cleaved Alk-SMase from the mucosa but also mildly increased the enzyme activity by ~50–70% in physiological concentrations. The enhancement was associated with its cleaving effect on the enzyme, because the activity of the COOH-terminal anchor truncated Alk-SMase was not increased by trypsin treatment. We previously demonstrated (14) that the activity of native Alk-SMase was not significantly changed after trypsin treatment. The Alk-SMase examined in the previous study was isolated from rat intestine; its activity may have already been enhanced by trypsin.

On the basis of this study, there are at least two forms of Alk-SMase: one is bound to the brush border with the COOH-terminal anchor, and the other is the free form of the enzyme in the intestinal lumen with the COOH-terminal anchor cleaved. Both forms are active, although the COOH-terminal truncated one has a higher activity. These enzymes are able to hydrolyze both endogenous and exogenous SM. The relative contribution of the two forms of Alk-SMase against different SM sources is not known. SM in the membrane is located in the out leaflet with the phosphocholine headgroup pointing outward. It is particularly abundant at the apical part of the microvillar (37, 38), where Alk-SMase is located (9). We hypothesize, therefore, that the membrane-bound Alk-SMase may predominantly hydrolyze membrane-bound SM and the free Alk-SMase in the intestinal lumen is the major enzyme targeting SM in the intestinal content. The hydrolysis of membrane SM may increase the concentration of ceramide, which may cause both physical and biological changes of the membrane, affecting cell proliferation and apoptosis, as reviewed by Kolesnick et al. (25). Hydrolysis of dietary SM in the lumen may increase luminal levels of ceramide. The generated ceramide could be further degraded by ceramidase to sphingosine, which can be absorbed readily and can play an antiproliferative role in the cells. We previously showed (11, 27, 32) the evidence of the presence of a specific ceramidase in the intestinal tract, and the distribution of intestinal ceramidase is parallel to that of Alk-SMase along the intestinal tract. Apart from ceramidase, it has been found that pancreatic bile salt- stimulated lipase has ceramidase-like activity (36). Thus pancreas can have an impact on hydrolysis of both SM and ceramide.

It was previously reported (21, 22, 39) that Alk-SMase activity was significantly decreased in the tissues of colonic cancer and longstanding ulcerative colitis. The mechanism for a reduced Alk-SMase in these tissues has not been established. We recently identified a transcriptional variant of the enzyme gene in colon cancer HT29 cells but not Caco-2 cells (42), indicating the mechanism leading to a reduced Alk-SMase in colonic tumors might be multiple. Overexpression of trypsin has been found to be associated with some colonic carcinomas, and the increased trypsin activity is correlated with the invasiveness of colon cancer (29, 43). Trypsin inhibitor was found to inhibit the tumorigenesis by ~40% in Min mice, a model of familial adenomatous polyposis (23). The promotive effect of trypsin on cancer progression was proposed to be related to degradation of extracellular matrix proteins (24) or specific cleavage and activation of protease-activated receptor-2 (5). Our findings in this article raise a question as to whether the increased trypsin activity in some colonic cancer tissues may cleave Alk-SMase and thereby affect tumorigenesis.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by grants from Swedish Cancer Foundation, Swedish Research Council, Albert Påhlsson foundation, Gunnar Nilsson Cancer Foundation, Swedish Medical Society, and the Foundation of Lund University Hospital.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R.-D. Duan, Gastroenterology Lab, Biomedical Center, B11 Lund Univ., S-221 84 Lund, Sweden (E-mail: Rui-dong.duan{at}med.lu.se)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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A. Nilsson and R.-D. Duan
Absorption and lipoprotein transport of sphingomyelin
J. Lipid Res., January 1, 2006; 47(1): 154 - 171.
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