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Enterokinase, the Initiator of Intestinal Digestion, Is a Mosaic Protease Composed of a Distinctive Assortment of Domains

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Enterokinase, the Initiator of Intestinal Digestion, Is a Mosaic Protease Composed of a Distinctive Assortment of Domains Proc. Natl. Acad. Sci. USA Vol. 91, pp. 7588-7592, August 1994 Biochemistry Enterokinase, the initiator of intestinal digestion, is a mosaic protease composed of a distinctive assortment of domains (serne proteases/tryngen activation) YASUNORI KITAMOTO*, XIN YUANt, QINGYU Wu*, DAVID W. MCCOURTt, AND J. EVAN SADLER*t* tHoward Hughes Medical Institute, *Departments of Medicine and Biochemistry and Molecular Biophysics, The Jewish Hospital of St. Louis, Washington University School of Medicine, St. Louis, MO 63110 Communicated by Earl W. Davie, April 19, 1994 ABSTRACT Enterokinase is a protease of the intestinal brates (7), except for the similar sequences of trypsinogens brush border that specifically cleaves the acidic propeptide from lungfish (IEEDK and LEDDK) and African clawed frog from trypsinogen to yield active trypsin. This cleavage initiates (FDDDK). Enterokinase prefers substrates with the se- a cascade of proteolytic reactions leading to the activation of quence DDDDK, whereas the presence ofaspartate residues many pancreatic zymogens. The full-length cDNA sequence for markedly inhibits the ability of trypsin to cleave such sub- bovine enterokinase and partial cDNA sequence for human strates (8). For example, toward bovine trypsinogen the enterokinase were determined. The deduced amino acid se- catalytic efficiency of enterokinase is 12,000-fold (porcine) quences indicate that active two-chain enterokinase is derived (9) or 34,000-fold (bovine) (10) greater than that of bovine from a single-chain precursor. Membrane association may be trypsin. This reciprocal specificity protects trypsinogen mediated by a potential sigal-anchor sequence near the amino against autoactivation by trypsin and promotes activation by terminus. The amino terminus of bovine enterokinase also enterokinase in the gut. meets the known sequence requirements for protein N-myris- Enterokinase has been purified from porcine (11), bovine toylation. The amino-terminal heavy chain contains domains (10, 12, 13), human (14), and ostrich intestine (15). With the that are homologous to segments of the low density lipoprotein possible exception of human enterokinase, which was sug- receptor, complement components COr and Cls, the macro- gested to be a heterotrimer (14), enterokinase appears to be phage scavenger receptor, and a recently described motif a disulfide-linked heterodimer with a heavy chain of 82-140 shared by the metalloprotease meprin and the Xenopus AS kDa and a light chain of 35-62 kDa. Mammalian enteroki- neuronal recognition protein. The carboxyl-terminal light nases contain 30-50%o carbohydrate, which may contribute to chain is homologous to the trypsin-like serine proteases. Thus, the apparent differences in polypeptide masses. The heavy enterokinase is a mosaic protein with a complex evolutionary chain is postulated to mediate association with the intestinal history. The amino acid sequence surrounding the amino brush border membrane (16), although no direct evidence for terminus of the enterokinase light chain is ITPK-IVGG (hu- this function has been reported. The light chain contains the man) or VSPK-IVGG (bovine), suggesting that single-chain center. Based enterokinase is activated by an unidentified trypsin-like pro- catalytic on susceptibility to inhibition by tease that cleaves the indicated Lys-fle bond. Therefore, en- chemical modification of the active-site serine and histidine terokinase may not be the "first" enzyme of the intestnal residues (9-11, 17) and on the partial amino acid sequence digestive hydrolase cascade. The specificity ofenterokinase for (18) and cDNA sequence of the bovine enterokinase light the DDDDK-I sequence of trpsinogen may be explained by chain (19), enterokinase is a member ofthe trypsin-like family complementary basic-amino acid residues clustered in poten- of serine proteases. tial S2-S5 subsites. Enterokinase stands at or near the top of a regulatory enzyme cascade that successfully limits the activity ofdiges- tive hydrolases to the gut, but there is no structural expla- All animals need to digest exogenous macromolecules with- nation for enterokinase membrane localization, substrate out destroying similar endogenous constituents. The regula- specificity, or expression specifically in the proximal small tion of digestive enzymes is, therefore, a fundamental re- intestine. To address these questions we have characterized quirement (1). Vertebrates have solved this problem, in part, cDNA clones for bovine and human by using a two-step enzymatic cascade to convert pancreatic enterokinase.§ zymogens to active enzymes in the lumen of the gut. The basic features ofthis cascade were described in 1899 by N. P. MATERIALS AND METHODS Schepovalnikov, working in the laboratory of I. P. Pavlov Materials. Purified calf enterokinase (EK-3, 131 units/4g) (2). Extracts of the proximal small intestine were shown to was strikingly activate the latent hydrolytic enzymes in pancre- from Biozyme Laboratories (San Diego). Fresh bovine atic fluid. Pavlov considered this intestinal factor to be an tissues were from a local abattoir. enzyme that activated other enzymes, or a "ferment of Amino Acid Sequencing. Enterokinase (16 pg) was reduced ferments," and named it "enterokinase." The importance of with 0.5% (vol/vol) 2-mercaptoethanol, separated by elec- this protease cascade is emphasized by the life-threatening trophoresis (20), transferred to an Immobilon P membrane intestinal malabsorption that accompanies congenital defi- (Millipore) by electroblotting, and stained with Coomassie ciency of enterokinase (3, 4). brilliant blue. The excised light-chain band (=47 kDa) was Enterokinase activates bovine trypsinogen by cleaving subjected to automated Edman degradation with an Applied after the sequence VDDDDK, releasing an amino-terminal Biosystems model 470A sequencer (21) equipped with a activation peptide (5, 6). The acidic DDDDK sequence ofthe model 120A phenylthiohydantoin analyzer. trypsinogen-activation peptide is conserved among verte- tTo whom reprint requests should be addressed at: Howard Hughes Medical Institute, Washington University, 660 South Euclid, Box The publication costs ofthis article were defrayed in part by page charge 8022, St. Louis, MO 63110. payment. This article must therefore be hereby marked "advertisement" §The sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession nos. U09859 and U09860). 7588 Downloaded by guest on September 27, 2021 Biochemistry: Kitamoto et aL Proc. NatL. Acad. Sci. USA 91 (1994) 7589 Isolation of cDNA Clones. RNA was extracted (22) from clones but absent in one (Fig. 1). This sequence is not bovine duodenum and proximal small intestine. Single- delimited by splice sites and therefore may be encoded by an stranded cDNA was prepared from total RNA (10 pg) using exon that is occasionally absent due to alternative splicing. avian myeloblastosis virus reverse transcriptase and an oli- This segment also could represent a length polymorphism. go(dT) primer (cDNA cycle kit, Invitrogen). The cDNA was The partial cDNA sequence for human enterokinase cor- used for PCR amplification (30 cycles of 2-min annealing at responds to amino acids 765-1035 encoded by the bovine 580C, 2-min extension at 720C, and 1-min denaturation at sequence. In the region of overlap, the open reading frames 940C) with sense primer 5'-TAY GAR GGI GCI TGG CCI of the bovine and human nucleotide sequences are -85% TGG GT-3' and antisense primer 5'-AAT GGG ACC GCC identical, and the encoded amino acid sequences are =84% IGA RTC ICC-3'. Products were analyzed by Southern identical. The 3' untranslated regions are less conserved, blotting and hybridization with 32P-labeled oligonucleotide exhibiting =67% sequence identity over 572 nt. probe 5'-STI WCI GCI GCC CAC TG-3'. The positive 572-bp By Northern blotting, an enterokinase mRNA species of product was cloned to yield pBEK1. =4.4 kb was detected in human small intestine, but not in Additional clones were obtained by radiolabeling the cDNA leukocytes, colon, ovary, testis, prostate, thymus, spleen, insert ofpBEK1 with [32P]dCTP (23) and screening ofbovine pancreas, kidney, skeletal muscle, liver, lung, placenta, or human small intestine Agtl1 cDNA libraries (Clontech) or brain, or heart (data not shown). This result is consistent with by using oligonucleotides to screen 5' rapid amplification of the studies of Pavlov on the distribution of enterokinase (2) cDNA ends (RACE) libraries (24). RACE libraries were and the immunohistochemical localization ofenterokinase in constructed with the 5' RACE system (GIBCO/BRL) using the brush border of duodenum and jejunum (27). bovine intestinal RNA and one of two sets of enterokinase- Structure of the Enterokinase Catalytic Domain. In agree- specific primers: set 1, 5'-TTA TTG TCT TCA TCA GAG ment with LaVallie et al. (19), amino acid residues 801-1035 CCA TC-3', 5'-TGG ACA GTT TAA TTC TCC ATC ACA-3', correspond to the enterokinase light chain, which has a 5'-ATC AAT TGC TAT GTA CTT TAG AGC-3'; set 2, predicted mass of 26.3 kDa, compared with 47 kDa observed 5'-ATT GAG ACA TTT CCT GTG ATA TCA ATG CTG-3', for purified bovine intestinal enterokinase (data not shown). 5'-TGT GGA AAG TGA CCA GTT GGC TGG ATT TAT-3', The difference reflects glycosylation ofthe light chain. There 5'-GCC TTG AAT CAG TTC TTC TT-3'. DNA sequences are three and four potential N-linked glycosylation sites, were determined on both strands (25). respectively,
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