Structure of murine enterokinase (enteropeptidase) and expression in small intestine during development

Xin Yuan, Xinglong Zheng, Deshun Lu, Deborah C. Rubin, Christopher Y. M. Pung, and J. Evan Sadler

Howard Hughes Medical Institute, Departments of Medicine and Biochemistry and Molecular Biophysics, Barnes-Jewish Hospital, Washington University School of Medicine, St. Louis, Missouri 63110

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Enterokinase (enteropeptidase) is expressed only in proximal small intestine, where it initiates digestive enzyme activation by converting trypsinogen into trypsin. To investigate this restricted expression pattern, mouse enterokinase cDNA was cloned, and the distribution of enterokinase mRNA and enzymatic activity were determined in adult mice and during gestation. Analysis of enterokinase sequences showed that a mucinlike domain near the NH2 terminus is composed of repeated ~15-amino acid Ser/Thr-rich motifs. By Northern blotting and trypsinogen activation assays, enterokinase mRNA and enzymatic activity were undetectable in stomach, abundant in duodenum, and decreased distally until they were undetectable in midjejunum, ileum, and colon. By in situ mRNA hybridization, enterokinase mRNA was localized to the enterocytes throughout the villus. Expression was not observed in goblet cells, Paneth cells, or Brunner's glands. Enterokinase mRNA and enzymatic activity were not detected in the duodenum of fetal mice but were easily detected in the duodenum on postnatal days 2-6. Both enterokinase mRNA and enzymatic activity decreased to very low levels after day 7 but increased after weaning and reached a high level characteristic of adult life by day 60. Therefore, in mice, duodenal enterocytes are the major type of cells expressing enterokinase, which appears to be regulated at the level of mRNA abundance.

duodenum; trypsinogen activation; serine protease; pancreatic enzymes

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ENTEROKINASE, also known as enteropeptidase, was discovered in Pavlov's laboratory as an activity of small intestinal extracts that stimulated the digestive enzymes of pancreatic secretions (33). Subsequent studies showed that enterokinase is a protease that activates trypsinogen (21). The resultant trypsin then cleaves and activates the zymogens of other pancreatic digestive hydrolases. Inherited deficiency of enterokinase impairs the initiation of this digestive hydrolase cascade, thereby causing intestinal malabsorption that may be life threatening (13, 14).

The amino acid sequences of bovine (19, 23), human (18, 19), porcine (29), and rat (39) enterokinase were recently determined indirectly by cDNA cloning. Enterokinase appears to be synthesized as a single-chain serine protease zymogen with an NH2-terminal hydrophobic domain that could mediate membrane association. This mechanism of membrane localization has not been demonstrated directly, and other models could account for the observed distribution of enterokinase.

In all animal species studied, enterokinase is highest in duodenum and rapidly decreases aborally, becoming undetectable by the distal jejunum (27, 32). In humans (1) and rats (24), this distribution is established late in fetal life. Enterokinase is associated with the brush border of enterocytes and appears to be absent in other cell types, although enterokinase antigen was reported in some goblet cells (16, 26, 30). Substantial quantities of free enterokinase also occur in mucinous secretions of bovine (10) and porcine (28) small intestine, suggesting that enterokinase could be secreted by other cells and localized secondarily on enterocytes.

To address the distribution and developmental regulation of enterokinase, the expression of enterokinase mRNA and enzymatic activity in mouse intestine was characterized in fetal and adult life. The complete cDNA sequence encoding murine enterokinase was cloned and employed to identify the cells within the proximal small intestine that express enterokinase mRNA by in situ hybridization.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Preparation of RNA. Total RNA was isolated from tissues of adult C57BL/6 mice (Jackson Laboratory) using RNAzol B (Biotex Laboratories, Houston, TX), according to the manufacturer's recommended procedures. Briefly, fresh tissue samples were frozen and stored in liquid nitrogen until use. Tissues were thawed on ice in RNAzol solution and homogenized with a Polytron model PT3000 homogenizer (Brinkmann) for <30 s at 2,400 rpm. Chloroform (0.1 vol) was added, and samples were incubated for 15 min on ice. After centrifugation at 14,000 g for 20 min, the upper layer was collected and reextracted with RNAzol B and chloroform. RNA was precipitated with ethanol and resuspended in water. Poly(A)+ mRNA was prepared from total RNA of mouse duodenum by oligo(dT) chromatography (Clontech) (3).

Isolation and characterization of mouse enterokinase cDNA. A pair of degenerate 20-nucleotide (nt) oligonucleotide primers was synthesized based on the conserved nucleotide sequence surrounding the active serine residue and the NH2-terminal light-chain sequence for bovine and human enterokinase (19). A fragment of mouse enterokinase cDNA was synthesized with these primers by reverse transcription-polymerase chain reaction (RT-PCR) using mouse duodenum total RNA as the template. The 576-bp product was cloned into TA cloning vector (Invitrogen) to generate plasmid pMEK1. For cDNA library screening, an EcoR I fragment containing the insert of pMEK1 was radiolabeled with [32P]dCTP by random oligonucleotide priming (9).

A cDNA library was prepared from 2 µg of duodenal poly(A)+ RNA using a ZAP Express cDNA synthesis kit (Stratagene). Five hundred thousand plaques of the library were screened with 32P-labeled pMEK1. Four positive clones were isolated, and corresponding pBK-CMV phagemid clones were generated by excision with helper phage. Clone MA7 contained a 4.1-kb insert with a 3.2-kb open reading frame and a poly(A) tail. Additional sequences at the 5'-end were cloned using the 5' rapid amplification of cDNA ends (RACE) system (GIBCO/BRL) with oligonucleotides corresponding to the 5'-end of clone MA7 as primers.

DNA sequences were determined on both strands by a dideoxy-chain termination method (36) using double-stranded templates (Sequenase 2.0, US Biochemical). Sequence analyses were performed with Lasergene software (DNASTAR).

Expression of recombinant murine enterokinase. A 5'-fragment of enterokinase cDNA was prepared by RT-PCR with poly(A)+ RNA of mouse duodenum and two oligonucleotide primers: 5'-primer from nt 20-46 with an engineered Nhe I site (underlined, changed residues in lowercase), CAC AGT GAA GAC Tg<UNL>C tAg C</UNL>AT TAG CTT G; 3'-primer from nt 619-639 (antisense), AGA GTG ACC AGT TGG CTG GAT. The product was digested with Nhe I and Bgl II and then cloned between the Nhe I and Bgl II sites at the 5'-end of clone MA7 in the vector pBK-CMV (Stratagene) to yield plasmid pMEK, encoding full-length mouse enterokinase under control of the CMV immediate early promoter. Plasmid pMEK was transfected into monkey COS-7 cells with lipofectamine (GIBCO/BRL Life Technologies) or into human 293T cells (8) with calcium phosphate. After 48 h, cell extracts and conditioned medium were assayed for enterokinase activity by activation of bovine trypsinogen (Worthington) as described (4).

Detection of enterokinase mRNA and enzymatic activity in gastrointestinal tract. Segments of gut were obtained from an adult C57BL/6 mouse. These included the stomach, between the gastroesophageal junction and pylorus; the duodenum, a segment of ~3.4-cm length between the pylorus and ligament of Treitz; jejunum, in four sequential ~3.4-cm segments (jejunum 1-4); and the colon, the proximal ~3.4-cm segment beginning at the ileocecal junction.

To collect tissue samples during gestation and postnatal development, adult C57BL/6 mice were mated at random, and female mice were examined every morning for vaginal plugs. Plugged mice were identified as embryonic day 0 and separated. The day of birth was defined as postnatal day 0. The entire small intestine was collected from mice on embryonic days 15 and 18 and postnatal days 0 (<6 h old) and 1 (6-24 h). Only duodenums were collected from postnatal day 2 onward. At each time point, the tissues from four animals were pooled and divided in equal portions for RNA isolation and enterokinase extraction.

Total RNA was prepared from each tissue sample with TRI-Reagent (Molecular Research Center, Cincinnati, OH). Samples of total RNA (20 µg) were electrophoresed on a 1.2% agarose gel containing 2.2 M formaldehyde and blotted onto nylon membrane (Gene Screen Plus, NEN) (40). Probes for Northern hybridization included a fragment (1,885 bp) corresponding to mouse enterokinase cDNA nt 400-2285 obtained by PCR, a 432-bp Dra II-Dra II fragment of a rat intestinal alkaline phosphatase cDNA clone (provided by M. J. Engle and D. H. Alpers, Washington University), and a 2-kb human beta -actin cDNA fragment (12). All probes were labeled with [32P]dCTP by random oligonucleotide priming (Rediprime, Amersham Life Science) (9).

Enterokinase was extracted from tissue samples (~200 mg tissue/1 ml buffer) by homogenization at 20,000 rpm for 1-2 min on ice (Polytron) in 20 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 8.0), 150 mM NaCl, and 1.0% Triton X-100. Enterokinase activity in the supernatant fraction was assayed as described (4) with minor modifications. Reactions (100 µl total vol) were incubated for 30 min at 25°C in wells of a microtiter plate containing tissue extract supernatant (20 µl), 125 nM trypsinogen, and 0.07 M sodium succinate (pH 6.5). The reaction was stopped by addition of 2 µl 2 M HCl, and the amount of trypsin generated was determined by adding 100 µl of 40 mM Tris · HCl (pH 8.0), 300 mM NaCl, 10 mM CaCl2, and 200 µM chromogenic substrate S-2765 (Kabi Pharmacia) to each reaction. Increase in absorbance at 405 nm was monitored, and the concentration of enterokinase was calculated based on comparison to a purified recombinant bovine enterokinase standard. The amount of enterokinase was normalized to protein concentration, which was quantitated with a micro-bicinchoninic acid kit (Pierce, Rockford, IL) with bovine serum albumin as a standard.

In situ hybridization analysis. A fragment containing nt 2702-3078 of mouse enterokinase cDNA was subcloned in the TA vector (Invitrogen) and linearized at the Xho I (antisense template) or Hind III site (sense template). Each template was used to transcribe in vitro single-stranded RNA with T7 RNA polymerase (antisense probe) or SP6 RNA polymerase (sense probe) labeled with [35S]UTP (NEN).

Tissues (adult duodenum) were fixed in 10% phosphate-buffered formaldehyde solution (Fisher) and embedded in paraffin. Sections (6-12 µm thick) were treated with xylene to remove paraffin, rehydrated in decreasing concentrations of ethanol (100 to 35%), and washed with phosphate-buffered saline (PBS). Sections were treated with proteinase K (1 µg/ml) for 30 min at 37°C, washed once with PBS, and acetylated for 10 min at room temperature in 0.25% acetic anhydride and 0.1 M triethanolamine HCl (pH 8.0). Sections were then washed twice in 2 × SSC [1 × SSC is 0.15 M NaCl-15 mM sodium citrate (pH 7.0)] and dehydrated with increasing concentrations of ethanol (35-100%). Sections were incubated in hybridization solution (50% formamide, 4 × SSC, 10% dextran sulfate, 375 µg/ml yeast tRNA, and 1 × Denhardt's solution containing 6 × 106 cpm/ml RNA probe) at 56°C for 15 h. Sections were then washed twice with 4 × SSC and digested with ribonuclease at 37°C for 1 h. This was followed by a series of 10-min washes at 37°C (4, 2, 0.5, and 0.1 × SSC, each containing 10 mM dithiothreitol). Postwashed slides were dipped in NTB-2 emulsion and exposed at 4°C for different times. At the end of each exposure, slides were processed in D19 developer and Rapid Fix (Kodak), stained with hematoxylin and eosin, and examined by phase-contrast and dark-field microscopy.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mouse enterokinase cDNA. A partial mouse enterokinase cDNA clone was prepared by RT-PCR, using primers based on conserved cDNA sequences of the human and bovine enterokinase serine protease domains. By hybridization with this probe, additional clones were obtained from a mouse duodenum cDNA library, and the remaining 5'-sequence was cloned by a 5'-RACE method. The composite sequence1 spans 4,215 nt and contains a large open reading frame between nucleotides 176 and 3,385. The ATG at position 176 is preceded by several in-frame stop codons and is in a favorable context to serve as an initiation codon (20). The stop codon (TAG) is followed by a 3'-untranslated sequence of 792 nt and a poly(A) tail. The open reading frame encodes a protein of 1,069 amino acids that is 70-88% identical in sequence to bovine (19, 23), human (18, 19), porcine (29), or rat (39) enterokinase. A full-length murine enterokinase cDNA was assembled and expressed by transient transfection of 293T cells and COS-7 cells; the recombinant protein activated bovine trypsinogen and was cell associated, confirming that the cloned sequence encoded enterokinase.

The sequence of murine enterokinase is highly conserved compared with enterokinase from other species, although there are some potentially significant differences. All of the repeated motifs found in other mammalian enterokinase proteins are present in murine enterokinase (Fig. 1). These include the two low-density lipoprotein receptor repeats (37), two complement component C1r/s repeats (25), one MAM domain (5), and one macrophage scavenger receptor domain (11). The serine protease domain is also highly conserved, including a sequence of four basic amino acids (Fig. 1, residues 919-922) proposed to interact with the acidic propeptide of the trypsinogen substrate (19, 29). There are two insertions in the serine protease domain of murine enterokinase. Four extra amino acids are inserted at positions 850-853, in a predicted surface loop that could interact with substrate residues on the COOH-terminal side of the scissile bond. A single alanine residue is inserted at position 971, in a surface loop below the substrate binding cleft. Both of these insertion sites are relatively variable among serine proteases (19).


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Fig. 1.   Amino acid sequence of murine proenterokinase. Translated composite cDNA sequence of mouse proenterokinase is aligned with sequences of rat, cow, human, and pig proenterokinase. Amino acid residues identical in all 4 sequences are boxed. Specific motifs are indicated by labeled brackets. These include proposed signal-anchor sequence and a potentially O-glycosylated mucinlike domain that is variable among different species. Four ~15 residue repeats within mucinlike domain are indicated and labeled a-d above aligned sequences. Other conserved motifs include 2 low-density lipoprotein receptor (LDLR) domains, 2 complement component C1r or C1s (C1r/s) domains, a MAM domain (named for motifs found in Meprin, Xenopus laevis A5 protein, and protein tyrosine phosphatase µ), and a macrophage scavenger receptor (MSCR) domain. Cysteine residues are shown in boldface. Potential N-linked glycosylation sites are indicated by underlined boldface type. Positions of activation cleavage site (arrow) between heavy and light chains and serine protease His, Asp, and Ser active site residues (bullet ) are indicated. A basic segment that is predicted to interact with acidic substrate residues is labeled (++++).

Some nonconserved features of the murine enterokinase heavy chain deserve comment. A potential N-myristoylation site is present at residue Gly2 in bovine, human and porcine enterokinase, and this glycine is replaced by Lys2 in murine enterokinase. The proposed signal-anchor segment of bovine, human, and porcine enterokinase contains a cysteine at position 32, and this is replaced by Ser32 in murine enterokinase. A more striking difference is present in the Ser/Thr-rich domain between residues 193-222. This segment is 30 amino acids longer in murine enterokinase than in bovine or porcine enterokinase and 45 amino acids longer than in human enterokinase. The additional residue appears to constitute imperfect copies of a 15-residue motif (Fig. 2). This domain of murine enterokinase also contains two potential N-glycosylation sites and a nonconserved cysteine residue. Finally, the MAM domain of murine enterokinase contains four additional cysteines that are not present in other MAM domains (Fig. 1); at present, there is no information on MAM domain structure that could help predict whether these cysteines might form intradomain disulfide bonds.


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Fig. 2.   Ser/Thr-rich repeats in enterokinase mucin domains. Approximately 15 amino acid mucinlike segments of mouse, rat, cow, human, and pig enterokinase were lettered (a-d) as shown in alignment of Fig. 1 and aligned using program Megalign (DNASTAR). Amino acid numbers are indicated at right. Residues matching consensus sequence shown at bottom are boxed and shaded. Consensus N-glycosylation sites and a cysteine residue in murine enterokinase are indicated in boldface.

Tissue and cellular expression of mouse enterokinase. The distribution of enterokinase expression in mouse intestine was determined by Northern blot (Fig. 3A). Enterokinase mRNA was detected as a 4.4-kb band. The levels of both mRNA and enzymatic activity were highest in duodenum and decreased in the aborad direction, becoming undetectable by the midjejunum (Fig. 3). For comparison, the blots were stripped and rehybridized with a probe for intestinal alkaline phosphatase, another brush-border enzyme that was found to be distributed throughout the jejunum. Neither enterokinase nor alkaline phosphatase was expressed in the stomach, ileum, or colon. Enterokinase mRNA also was not detected in the pancreas, liver, testis, or brain of adult mice (data not shown).


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Fig. 3.   Tissue distribution of mouse enterokinase mRNA and enzymatic activity. A: total cellular RNA was extracted from segments of mouse gastrointestinal tract and analyzed by Northern blotting. Membrane was hybridized sequentially with probes for mouse enterokinase (top), rat intestinal alkaline phosphatase (IAP), and human beta -actin. B: enterokinase enzyme activity was assayed in detergent extracts of tissue segment as described under MATERIALS AND METHODS. Values are expressed as enterokinase (ng) relative to amount of total protein (mg) in extract.

Enterokinase expression along the villus-crypt axis was examined by in situ RNA hybridization with radiolabeled enterokinase single-stranded RNA probes (Fig. 4). When hybridized with the antisense probe, sections of duodenum demonstrated many autoradiographic grains over the villi. The crypt cells did not have overlying grains in a concentration significantly greater than background. Under higher bright-field magnification, in well-oriented 608-µm sections, the presence of grains was determined for 200 enterocytes and 50 goblet cells, and grains were seen only over the cytoplasm of enterocytes. No grains were detected over the rare Paneth cells, and no specific hybridization was detected at the level of Brunner's glands. Tissue sections hybridized with the control sense single-stranded RNA probe did not exhibit hybridization signals.


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Fig. 4.   In situ mRNA hybridization analysis of mouse enterokinase in duodenal tissues. Sections of adult mouse duodenum were hybridized with 35S-labeled RNA probes for enterokinase (magnification: ×125, A and B; ×160, C and D). A: antisense probe, 10- to 12-µm section. B: same section as in A with bright-field illumination. C: antisense probe, 6- to 8-µm section. D: control sense probe, 6- to 8-µm section.

Developmental expression of mouse enterokinase. Enterokinase expression in the duodenum during embryonic and postnatal development was evaluated by Northern blotting and enzymatic assays (Fig. 5). Enterokinase mRNA and activity were not detected before birth. Enterokinase activity was highly expressed within the first 24 h of life, at a time when enterokinase mRNA was barely detectable. Enterokinase mRNA levels peaked during postnatal days 2-4, declined to very low levels from postnatal days 6 to 12, became easily detected by Northern blotting by postnatal day 21, and reached a high level of expression characteristic of adult life by postnatal day 60. The more sensitive RT-PCR method demonstrated the presence of enterokinase mRNA on postnatal days 0 and 8-12 (data not shown). Enterokinase activity tended to follow enterokinase mRNA levels but appeared to be more sensitive; activity was always readily detected in the postnatal period, and an increase in activity was observed on postnatal day 12 before the increase in mRNA expression was apparent by Northern blotting on postnatal day 21. The timing of this later increase was somewhat variable; another cohort of mice showed a rise in enterokinase mRNA to the adult level by postnatal day 16 (data not shown). In contrast, mRNA for intestinal alkaline phosphatase could be detected on postnatal day 0, and the level remained relatively constant throughout postnatal life (Fig. 5A).


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Fig. 5.   Developmental expression of enterokinase in duodenum. A: total cellular RNA was extracted from mouse small intestine at indicated embryonic dates (E) and from mouse duodenum at different postnatal dates (D) and analyzed by Northern blot as described under MATERIALS AND METHODS. Membrane was sequentially hybridized with probes for mouse enterokinase (top), rat IAP, and human beta -actin, respectively. B: enterokinase activity was determined in duodenal tissue extracts of mice during embryonic and postnatal stages.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Several features of the murine enterokinase sequence provide new insights into the structure-function relationships of this protease. The NH2-terminal amino acid sequences of bovine, human, and porcine enterokinase appear to meet the substrate specificity requirements for N-myristoyltransferase (35), suggesting that Gly2 may be myristoylated. However, Gly2 is not conserved in murine or rat enterokinase (39), indicating that myristoylation is not generally necessary for enterokinase biosynthesis or membrane localization.

Enterokinase from several species was reported to have a segment of variable length, beginning at amino acid residue ~166, which is rich in Ser and Thr residues (18, 19, 29, 39) and may be O-glycosylated (29). One bovine enterokinase clone lacked the entire segment, which would be consistent with additional length polymorphism or alternative splicing (19). Examination of the longer murine enterokinase sequence (Fig. 1) indicates that, in all species, this segment is composed of tandem repeats of an ~15-amino acid motif (Fig. 2). Similar repeats are characteristic of heavily O-glycosylated epithelial mucins. For example, human small intestinal mucin MUC2 contains ~100 repeats of a 23-amino acid sequence, and this tandem array apparently is not divided by introns within the MUC2 gene (38). The frog integumentary mucin FIM-B.1 contains at least 22 repeats of an 11-residue sequence, but each of these repeats is encoded by a separate exon and alternative splicing yields a polydisperse family of FIM-B.1 mRNA species (34). The enterokinase mucin-type repeats are distinctive because some contain consensus sites for N-glycosylation, and one murine enterokinase repeat contains a cysteine (Fig. 2). The function of the variable mucin domain in enterokinase is not known, although extensive O-linked glycosylation may protect enterokinase from proteolysis.

The cell type in intestine that produces enterokinase has been controversial. Both enterokinase activity and antigen are associated with the brush border of enterocytes in the duodenum and proximal jejunum (16, 26, 30), although enterokinase antigen was reported in occasional goblet cells (30). By Northern blotting, enterokinase mRNA appears to be restricted to the small intestine (18). This pattern would be consistent with the synthesis of enterokinase by enterocytes and localization therein as an integral protein of the microvillus membrane. However, substantial quantities of free enterokinase appear to occur in the mucinous secretions of bovine (10) and porcine (28) small intestine. Furthermore, purified porcine enterokinase was found to lack the predicted NH2-terminal transmembrane domain, presumably due to proteolytic processing (29), suggesting that enterokinase could be synthesized and secreted by other cell types and localized secondarily in the brush border of enterocytes. In that case, the observed proximal-distal gradient of enterokinase in the small intestine would also not necessarily reflect the pattern of synthesis, and localization of enterokinase in jejunum, for example, might be due to transport from the duodenum. However, studies of mRNA localization indicate that the sites of enterokinase synthesis and activity in vivo do correspond. Enterokinase mRNA and enzymatic acitivity are present in duodenum and proximal jejunum (Fig. 3), and the results of in situ mRNA hybridization (Fig. 4) appear to exclude cells other than enterocytes as major sources of enterokinase.

Enterokinase expression during fetal and postnatal development has previously been studied only by enzymatic activity assays. In the rat, enterokinase activity appeared at embryonic day 20 and rapidly increased to the adult level by postnatal day 2 (24). In the mouse, we found a burst of enterokinase activity in the duodenum on postnatal day 0 and lower activity during the following 3 wk of life, after which enterokinase increased to the adult level (Fig. 5B); this pattern is consistent with a previous report (2). The appearance of enterokinase activity at the time of birth coincides with a major step in the maturation of small intestinal epithelium, the development of crypts and restriction of cell proliferation to stem cells within them (7, 17). The later increase corresponds approximately to the time of weaning and also is accompanied by functional changes in the intestinal epithelium to accommodate the adult diet. A similar pattern has been reported for the activities of several other brush-border hydrolases, including sucrase-isomaltase and maltase (15). The mechanisms that regulate the expression of these genes are not fully characterized.

Enterokinase mRNA appeared to parallel these changes in enzyme activity, indicating that enterokinase is regulated at least by mRNA abundance, possibly at the level of transcription. However, the correlation of mRNA and activity was imperfect. For example, substantial enterokinase activity was present at times when mRNA levels were low or undetectable by Northern blotting, especially on postnatal day 0 and between postnatal days 8 and 12 (Fig. 5). This discrepancy may reflect a higher sensitivity of enterokinase activity assays compared with Northern blotting, since enterokinase mRNA was detected easily at all of these time points by RT-PCR. Alternatively, enterokinase may also be regulated at a posttranscriptional level such as translation or posttranslational modification.

The mRNA and enzyme activity of enterokinase restricted mainly in duodenum and proximal jejunum suggests that enterokinase gene expression was controlled by a promoter whose properties could provide insight into the regional specialization of enterocytes during development. In addition, enterokinase is regulated dramatically in mature duodenum by pancreatic secretions. An isolated loop of dog intestine lost enterokinase activity after several hours, and addition of pancreatic juice restored enterokinase within 30 min; this stimulatory activity of pancreatic juice was destroyed by boiling (33). Similar results were obtained in a mouse model of exocrine pancreatic deficiency: enterokinase activity was nearly absent from the duodenum of CBA/J-epi mice but was induced to normal levels by feeding trypsinogen (22). The dependence of enterokinase activity on contact with pancreatic secretions also has been demonstrated in rat (31) and guinea pig models (6). Additional study will be required to determine whether luminal contents regulate enterokinase at the level of transcription, mRNA stability or translation, or zymogen activation.

    ACKNOWLEDGEMENTS

The authors thank Dr. Qingyu Wu (Berlex Biosciences) and Dr. Yasunori Kitamoto (Kumamoto University) for helpful discussions; Drs. David H. Alpers and Michael J. Engle (Washington University) for the rat intestinal alkaline phosphatase probe; Cathy Gapart, Jill Robey, Teresa Tolley, and Qingmei Xie (Washington University) for assistance with in situ mRNA hybridization assays; and Lisa Westfield for animal breeding.

    FOOTNOTES

These studies were supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-50053 (to J. E. Sadler) and DK-46122 (to D. C. Rubin).

1 The mouse enteropeptidase cDNA sequence is in the GenBank database under Accession No. U73378.

Address for reprint requests: J. E. Sadler, Howard Hughes Medical Institute, Washington University School of Medicine, 660 South Euclid Ave., Box 8022, St. Louis, MO 63110.

Received 15 October 1996; accepted in final form 21 October 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Antonowicz, I., and E. Lebenthal. Developmental pattern of small intestinal enterokinase and disaccharidase activities in the human fetus. Gastroenterology 72: 1299-1303, 1977[Medline].

2.   Arsenault, P., and D. Menard. Development of enteropeptidase activity in mouse small intestine: influence of hormones. Can. J. Physiol. Pharmacol. 63: 472-475, 1985[Medline].

3.   Aviv, H., and P. Leder. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA 69: 1408-1412, 1972[Abstract].

4.   Baratti, J., S. Maroux, and D. Louvard. Effect of ionic strength and calcium ions on the activation of trypsinogen by enterokinase. Biochim. Biophys. Acta 321: 632-638, 1973[Medline].

5.   Beckmann, G., and P. Bork. An adhesive domain detected in functionally diverse receptors. Trends Biochem. Sci. 18: 40-41, 1993[Medline].

6.   Bett, N. J. Regulation of enterokinase synthesis in animal and human small intestine by luminal signals: its implication in upper gastrointestinal surgery. Br. J. Surg. 66: 708-711, 1979[Medline].

7.   Calvert, R., and P. Pothier. Migration of fetal intestinal intervillous cells in neonatal mice. Anat. Rec. 227: 199-206, 1990[Medline].

8.   Dubridge, R. B., P. Tang, H. S. Hsia, P.-M. Leong, J. H. Miller, and M. P. Calos. Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol. Cell. Biol. 7: 379-387, 1987[Medline].

9.   Feinberg, A. P., and B. Vogelstein. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity (Addendum). Anal. Biochem. 137: 266-267, 1984[Medline].

10.   Fonseca, P., and P. Light. The purification and characterization of bovine enterokinase from membrane fragments in the duodenal mucosal fluid. J. Biol. Chem. 258: 14516-14520, 1983[Abstract/Free Full Text].

11.   Freeman, M., J. Ashenas, D. J. G. Rees, D. M. Kingsley, N. G. Copeland, N. A. Jenkins, and M. Krieger. An ancient, highly conserved family of cysteine-rich protein domains revealed by cloning type I and type II murine macrophage scavenger receptors. Proc. Natl. Acad. Sci. USA 87: 8810-8814, 1990[Abstract].

12.   Gunning, P., P. Ponte, H. Okayama, J. Engel, H. Blau, and L. Kedes. Isolation and characterization of full-length cDNA clones for human alpha -, beta -, and gamma -actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. Mol. Cell. Biol. 3: 787-795, 1983[Medline].

13.   Hadorn, B., M. J. Tarlow, J. K. Lloyd, and O. H. Wolff. Intestinal enterokinase deficiency. Lancet 1: 812-813, 1969[Medline].

14.   Haworth, J. C., B. Gourley, B. Hadorn, and C. Sumida. Malabsorption and growth failure due to intestinal enterokinase deficiency. J. Pediatr. 78: 481-490, 1971[Medline].

15.   Henning, S. J. Functional development of the gastrointestinal tract. In: Physiology of the Gastrointestinal Tract (2nd ed.), edited by L. R. Johnson. New York: Raven, 1987, p. 285-300.

16.   Hermon-Taylor, J., J. Perrin, D. A. W. Grant, A. Appleyard, and A. I. Magee. Immunofluorescent localization of enterokinase in human small intestine. Gut 18: 259-265, 1977[Abstract].

17.   Hermos, J. A., M. Mathan, and J. S. Trier. DNA synthesis and proliferation by villous epithelial cells in fetal rats. J. Cell Biol. 50: 255-258, 1971[Free Full Text].

18.   Kitamoto, Y., R. A. Veile, H. Donis-Keller, and J. E. Sadler. cDNA sequence and chromosomal localization of human enterokinase, the proteolytic activator of trypsinogen. Biochemistry 34: 4562-4568, 1995[Medline].

19.   Kitamoto, Y., X. Yuan, Q. Wu, D. W. McCourt, and J. E. Sadler. Enterokinase, the initiator of intestinal digestion, is a mosaic protease composed of a distinctive assortment of domains. Proc. Natl. Acad. Sci. USA 91: 7588-7592, 1994[Abstract].

20.   Kozak, M. An analysis of vertebrate mRNA sequences: intimations of translational control. J. Cell Biol. 115: 887-903, 1991[Abstract].

21.   Kunitz, M. Formation of trypsin from crystalline trypsinogen by means of enterokinase. J. Gen. Physiol. 22: 429-446, 1939[Free Full Text].

22.   Kwong, W. K. L., B. Seetharam, and D. H. Alpers. Effect of exocrine pancreatic insufficiency on small intestine in the mouse. Gastroenterology 74: 1277-1282, 1978[Medline].

23.   LaVallie, E. R., A. Rehemtulla, L. A. Racie, E. A. DiBlasio, C. Ferenz, K. L. Grant, A. Light, and J. M. McCoy. Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase. J. Biol. Chem. 268: 23311-23317, 1993[Abstract/Free Full Text].

24.   Lebenthal, E. Induction of fetal rat enterokinase (enteropeptidase EC 3.4.21.9) in utero by hydrocortisone and actinomycin D. Pediatr. Res. 11: 282-285, 1977[Abstract].

25.   Leytus, S. P., K. Kurachi, K. S. Sakariassen, and E. W. Davie. Nucleotide sequence of the cDNA coding for human complement C1r. Biochemistry 25: 4855-4863, 1986[Medline].

26.   Lojda, Z., and R. Gossrau. Histochemical demonstration of enteropeptidase activity. New method with a synthetic substrate and its comparison with the trypsinogen procedure. Histochemistry 78: 251-270, 1983[Medline].

27.   Lojda, Z., and F. Malis. Histochemical demonstration of enterokinase. Histochemie 32: 23-29, 1972[Medline].

28.   Louvard, D., S. Marous, J. Baratti, and P. Desnuelle. On the distribution of enterokinase in porcine intestine and on its subcellular localization. Biochim. Biophys. Acta 309: 127-137, 1973[Medline].

29.   Matsushima, M., M. Ichinose, N. Yahagi, N. Kakei, S. Tsukada, K. Miki, K. Kurokawa, K. Tashiro, K. Shiokawa, K. Shinomiya, H. Umeyama, H. Inoue, T. Takahashi, and K. Takahashi. Structural characterization of porcine enteropeptidase. J. Biol. Chem. 269: 19976-19982, 1994[Abstract/Free Full Text].

30.   Miyoshi, Y., T. Onishi, T. Sano, and N. Komi. Monoclonal antibody against human enterokinase and immunohistochemical localization of the enzyme. Gastroenterol. Jpn. 25: 320-327, 1990[Medline].

31.   Newman, B. M., P. C. Lee, H. Tajiri, D. R. Cooney, and E. Lebenthal. Pancreaticobiliary factors in the modulation of small intestinal enterokinase in the rat. Am. J. Physiol. 250 (Gastrointest. Liver Physiol. 13): G103-G108, 1986[Medline].

32.   Nordstrom, C., and A. Dahlqvist. Rat enterokinase: the effect of ions and the localization in the intestine. Biochim. Biophys. Acta 242: 209-225, 1971[Medline].

33.   Pavlov, I. P. The Work of the Digestive Glands, translated by W. H. Thompson. London: Griffin, 1902, p. 148-163.

34.   Probst, J. C., F. Hauser, W. Joba, and W. Hoffmann. The polymorphic integumentary mucin B.1 from Xenopus laevis contains the short consensus repeat. J. Biol. Chem. 267: 6310-6316, 1992[Abstract/Free Full Text].

35.   Rudnick, D. A., C. A. McWherter, G. W. Gokel, and J. I. Gordon. Myristoyl-CoA:protein B-myristoyltransferase. Adv. Enzymol. 67: 375-430, 1993.[Medline]

36.   Sanger, F., S. Nicklen, and A. R. Coulson. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467, 1977[Abstract].

37.   Sudhof, T. C., J. L. Goldstein, M. S. Brown, and D. W. Russell. The LDL receptor gene: a mosaic of exons shared with different proteins. Science 228: 815-822, 1985[Medline].

38.   Toribara, N. W., J. R. Gum, Jr., P. J. Culhane, R. E. Lagace, J. W. Hicks, G. M. Petersen, and Y. S. Kim. MUC-2 human small intestinal mucin gene structure. Repeated arrays and polymorphism. J. Clin. Invest. 88: 1005-1013, 1991[Medline].

39.   Yahagi, N., M. Ichinose, M. Matsushima, Y. Matsubara, K. Miki, K. Kurokawa, H. Fukamachi, K. Tashiro, K. Shiokawa, T. Kageyama, T. Takahashi, H. Inoue, and K. Takahashi. Complementary DNA cloning and sequencing of rat enteropeptidase and tissue distribution of its mRNA. Biochem. Biophys. Res. Commun. 219: 806-812, 1996[Medline].

40.   Zheng, X. L., and W. J. Hendry. Neonatal diethylstilbestrol treatment alters the estrogen-regulated expression of both cell proliferation and apoptosis-related proto-oncogenes (c-jun, c-fos, c-myc, bax, bcl-2, and bcl-x) in the hamster uterus. Cell Growth Differ. 8: 425-434, 1997[Abstract].


AJP Gastroint Liver Physiol 274(2):G342-G349
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