Identification of a Novel Acidic Mammalian Chitinase Distinct from Chitotriosidase
2001; Elsevier BV; Volume: 276; Issue: 9 Linguagem: Inglês
10.1074/jbc.m009886200
ISSN1083-351X
AutoresRolf G. Boot, Edward F. C. Blommaart, Erwin Swart, Karen Ghauharali‐van der Vlugt, Nora Bijl, Cassandra Moe, Allen R. Place, Johannes M. F. G. Aerts,
Tópico(s)Insect symbiosis and bacterial influences
ResumoChitinases are ubiquitous chitin-fragmenting hydrolases. Recently we discovered the first human chitinase, named chitotriosidase, that is specifically expressed by phagocytes. We here report the identification, purification, and subsequent cloning of a second mammalian chitinase. This enzyme is characterized by an acidic isoelectric point and therefore named acidic mammalian chitinase (AMCase). In rodents and man the enzyme is relatively abundant in the gastrointestinal tract and is found to a lesser extent in the lung. Like chitotriosidase, AMCase is synthesized as a 50-kDa protein containing a 39-kDa N-terminal catalytic domain, a hinge region, and a C-terminal chitin-binding domain. In contrast to chitotriosidase, the enzyme is extremely acid stable and shows a distinct second pH optimum around pH 2. AMCase is capable of cleaving artificial chitin-like substrates as well as crab shell chitin and chitin as present in the fungal cell wall. Our study has revealed the existence of a chitinolytic enzyme in the gastrointestinal tract and lung that may play a role in digestion and/or defense. Chitinases are ubiquitous chitin-fragmenting hydrolases. Recently we discovered the first human chitinase, named chitotriosidase, that is specifically expressed by phagocytes. We here report the identification, purification, and subsequent cloning of a second mammalian chitinase. This enzyme is characterized by an acidic isoelectric point and therefore named acidic mammalian chitinase (AMCase). In rodents and man the enzyme is relatively abundant in the gastrointestinal tract and is found to a lesser extent in the lung. Like chitotriosidase, AMCase is synthesized as a 50-kDa protein containing a 39-kDa N-terminal catalytic domain, a hinge region, and a C-terminal chitin-binding domain. In contrast to chitotriosidase, the enzyme is extremely acid stable and shows a distinct second pH optimum around pH 2. AMCase is capable of cleaving artificial chitin-like substrates as well as crab shell chitin and chitin as present in the fungal cell wall. Our study has revealed the existence of a chitinolytic enzyme in the gastrointestinal tract and lung that may play a role in digestion and/or defense. acidic mammalian chitinase 4-methylumbelliferyl β-d-N,N′-diacetylchitobiose 4-morpholineethanesulfonic acid polyacrylamide gel electrophoresis expressed sequence tag polymerase chain reaction Next to cellulose, chitin is the most abundant glycopolymer on earth, being present as a structural component in coatings of many species, such as the cell wall of most fungi (1Debono M. Gordee R.S. Annu. Rev. Microbiol. 1994; 48: 471-497Crossref PubMed Scopus (363) Google Scholar), the microfilarial sheath of parasitic nematodes (2Fuhrman J.A. Piessens W.F. Mol. Biochem. Parasitol. 1985; 17: 93-104Crossref PubMed Scopus (85) Google Scholar, 3Araujo A.C. Souto-Padron T. de Souza W.J. Histo. Cyto. 1993; 41: 571-578Crossref PubMed Scopus (67) Google Scholar), and the exoskeleton of all types of arthropods (4Neville A.C. Parry D.A. Woodhead-Galloway J. J. Cell Sci. 1976; 21: 73-82Crossref PubMed Google Scholar), and in the lining of guts of many insects (5Shahabuddin M. Kaslow D.C. Exp. Parasit. 1994; 79: 85-88Crossref PubMed Scopus (71) Google Scholar). Chitinases (EC 3.2.1.14) are endo-β-1,4-N-acetylglucosaminidases that can fragment chitin and have been identified in several organisms (6Flach J. Pilet P.E. Jolles P. Experientia. 1992; 48: 701-716Crossref PubMed Scopus (332) Google Scholar). Until a few years ago it was generally assumed that man lacks the ability to produce a functional chitinase. Our observation of a markedly elevated chitotriosidase activity in plasma of symptomatic Gaucher patients formed the basis for the subsequent identification of a human phagocyte-specific chitinase, named chitotriosidase (7Hollak C.E.M. van Weely S. van Oers M.H.J. Aerts J.M.F.G. J. Clin. Invest. 1994; 93: 1288-1292Crossref PubMed Scopus (748) Google Scholar, 8Renkema G.H. Boot R.G. Muijsers A.O. Donker-Koopman W.E. Aerts J.M.F.G. J. Biol. Chem. 1995; 270: 2198-2202Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 9Boot R.G. Renkema G.H. Strijland A. van Zonneveld A.J. Aerts J.M.F.G. J. Biol. Chem. 1995; 270: 26252-26256Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Tissue macrophages can synthesize large amounts of chitotriosidase upon an appropriate stimulus, such as the massive lysosomal lipid accumulation that occurs in macrophages of Gaucher patients (7Hollak C.E.M. van Weely S. van Oers M.H.J. Aerts J.M.F.G. J. Clin. Invest. 1994; 93: 1288-1292Crossref PubMed Scopus (748) Google Scholar). Chitotriosidase is largely secreted as a 50-kDa active enzyme containing a C-terminal chitin binding domain (10Renkema G.H. Boot R.G. Strijland A. Donker-Koopman W.E. van den Berg M. Muijsers A.O. Aerts J.M.F.G. Eur. J. Biochem. 1997; 244: 279-285Crossref PubMed Scopus (150) Google Scholar, 11Tjoelker L.W. Gosting L. Frey S. Hunter C.L. Trong H.L. Steiner B. Brammer H. Gray P.W. J. Biol. Chem. 2000; 275: 514-520Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In macrophages some enzyme is proteolytically processed to a C-terminally truncated 39-kDa form with hydrolase activity that accumulates in lysosomes of these cells (10Renkema G.H. Boot R.G. Strijland A. Donker-Koopman W.E. van den Berg M. Muijsers A.O. Aerts J.M.F.G. Eur. J. Biochem. 1997; 244: 279-285Crossref PubMed Scopus (150) Google Scholar). The 50-kDa chitotriosidase form is also synthesized by progenitors of neutrophilic granulocytes (9Boot R.G. Renkema G.H. Strijland A. van Zonneveld A.J. Aerts J.M.F.G. J. Biol. Chem. 1995; 270: 26252-26256Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar) and stored in their specific granules (9Boot R.G. Renkema G.H. Strijland A. van Zonneveld A.J. Aerts J.M.F.G. J. Biol. Chem. 1995; 270: 26252-26256Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar,12Boussac M. Garin J. Electrophoresis. 2000; 21: 665-672Crossref PubMed Scopus (111) Google Scholar). Chitotriosidase is remarkably homologous to chitinases from plants, bacteria, fungi, nematodes and insects (8Renkema G.H. Boot R.G. Muijsers A.O. Donker-Koopman W.E. Aerts J.M.F.G. J. Biol. Chem. 1995; 270: 2198-2202Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 9Boot R.G. Renkema G.H. Strijland A. van Zonneveld A.J. Aerts J.M.F.G. J. Biol. Chem. 1995; 270: 26252-26256Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Analogous to some plant chitinases, recombinant chitotriosidase has been found to inhibit hyphal growth of chitin-containing fungi such as Candida andAspergillusspecies. 1R. G. Boot, E. F. C. Blommaart, G. W. Gooday, B. A. Friedman, and J. M. F. G. Aerts, manuscript in preparation. 1R. G. Boot, E. F. C. Blommaart, G. W. Gooday, B. A. Friedman, and J. M. F. G. Aerts, manuscript in preparation.The specific expression by phagocytes also suggests a physiological role in defense against chitin-containing pathogens. A recessively inherited deficiency in chitotriosidase activity is frequently encountered (7Hollak C.E.M. van Weely S. van Oers M.H.J. Aerts J.M.F.G. J. Clin. Invest. 1994; 93: 1288-1292Crossref PubMed Scopus (748) Google Scholar, 13Guo Y.F. He W. Boer A.M. Wevers R.A. Debruijn A.M. Groener J.E.M.M. Hollak C.E.M. Aerts J.M.F.G. Galjaard H. Van Diggelen O.P. J. Inher. Metab. Dis. 1995; 18: 717-722Crossref PubMed Scopus (179) Google Scholar). About 1 in 20 individuals is completely deficient in enzymatically active chitotriosidase, because of a 24-base pair duplication in the chitotriosidase gene (14Boot R.G. Renkema G.H. Verhoek M. Strijland A. Bliek J. de Meulemeester T.M. Mannens M.M. Aerts J.M. J. Biol. Chem. 1998; 273: 25680-25685Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). This duplication, which occurs panethnically, leads to strongly reduced amounts of an abnormally spliced mRNA only, encoding an enzymatically inactive protein that lacks an internal stretch of 29 amino acids (14Boot R.G. Renkema G.H. Verhoek M. Strijland A. Bliek J. de Meulemeester T.M. Mannens M.M. Aerts J.M. J. Biol. Chem. 1998; 273: 25680-25685Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). In Caucasian populations, up to 35‥ of all individuals carry this abnormal chitotriosidase allele and about 5‥ are homozygous for this allele (14Boot R.G. Renkema G.H. Verhoek M. Strijland A. Bliek J. de Meulemeester T.M. Mannens M.M. Aerts J.M. J. Biol. Chem. 1998; 273: 25680-25685Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). The prevalence of deficiency suggests that chitotriosidase no longer fulfills an important defense function under normal circumstances or, alternatively, that other mechanisms may compensate the lack of functional chitotriosidase. To test whether compensatory mechanisms exist, we have searched for other chitinases in mammals. The discovery of a second mammalian chitinolytic enzyme is described here. The properties of this acidic mammalian chitinase (AMCase)2are reported, and the possible implications of its existence are discussed. Chitinase enzyme activity was determined with the fluorogenic substrates 4-methylumbelliferyl β-d-N,N′-diacetylchitobiose (4MU-chitobiose; Sigma) and 4-methylumbelliferyl β-d-N,N′,N“-triacetylchitotriose (Sigma). Assay mixtures contained 0.027 mm substrate and 1 mg/ml of bovine serum albumin in McIlvaine buffer (100 mmcitric acid, 200 mm sodium phosphate) at the indicated pH. The standard enzyme activity assay for human chitotriosidase with 4-methylumbelliferyl β-d-N,N′,N”-triacetylchitotriose substrate was performed at pH 5.2, as previously described (7Hollak C.E.M. van Weely S. van Oers M.H.J. Aerts J.M.F.G. J. Clin. Invest. 1994; 93: 1288-1292Crossref PubMed Scopus (748) Google Scholar). The standard AMCase enzyme activity assays with 4MU-chitobiose substrate were performed at pH 4.5. Crab shell chitin (Poly- (1Debono M. Gordee R.S. Annu. Rev. Microbiol. 1994; 48: 471-497Crossref PubMed Scopus (363) Google Scholar, 2Fuhrman J.A. Piessens W.F. Mol. Biochem. Parasitol. 1985; 17: 93-104Crossref PubMed Scopus (85) Google Scholar, 3Araujo A.C. Souto-Padron T. de Souza W.J. Histo. Cyto. 1993; 41: 571-578Crossref PubMed Scopus (67) Google Scholar, 4Neville A.C. Parry D.A. Woodhead-Galloway J. J. Cell Sci. 1976; 21: 73-82Crossref PubMed Google Scholar)-β-d-N-acetylglucosamine, Sigma) was used as a natural substrate to determine chitinase activity as described (10Renkema G.H. Boot R.G. Strijland A. Donker-Koopman W.E. van den Berg M. Muijsers A.O. Aerts J.M.F.G. Eur. J. Biochem. 1997; 244: 279-285Crossref PubMed Scopus (150) Google Scholar). The chitin fragments were analyzed by fluorophore-assisted carbohydrate electrophoresis as described by Jackson (15Jackson P. Biochem. J. 1990; 270: 705-713Crossref PubMed Scopus (285) Google Scholar). Measurements of chitin formation during regeneration of fungal spheroplasts was performed as described by Hector and Braun (16Hector R.F. Braun P.C. J. Clin. Microbiol. 1986; 24: 620-624Crossref PubMed Google Scholar). Briefly, spheroplasts were prepared from the Candida albicans strain CAi-4 (ura3), grown overnight in YPD medium (1‥ yeast extract, 2‥ peptone, 2‥ glucose) at 28 °C. Cells were concentrated by centrifugation and incubated with 2.5 mg/ml zymolyase (100T, ICN Immuno Biologicals, Costa Mesa, CA) in buffer containing 50 mmsodium phosphate, pH 7.5, 1.2 m sorbitol, and 27 mm β-mercaptoethanol for 60 min at 37 °C. After extensive washing, spheroplasts were allowed to regenerate in 96-well microtiter plates in regeneration buffer (0.25‥ (w/v) MES buffer, pH 6.7, containing 0.17‥ (w/v) yeast nitrogen base (without amino acids and ammonium sulfate; Sigma), 0.15‥ (w/v) ammonium sulfate, 2‥ (w/v) glucose, 1.2 m sorbitol, 20 μg/ml uridine) at 37 °C. Chitinase enzyme preparations were added to a final concentration of 3 μg/ml. After a 2-h incubation, 50 μl of 300 μg/ml Calcofluor white (Sigma) in 10 mm sodium phosphate buffer, pH 7.5, containing 1.2 m sorbitol was added. After 5 min the plates were washed with buffer only, and fluorescence was determined using a LS 50 Perkin Elmer fluorimeter (excitation, 405 nm; emission, 450 nm). Detergent-free extracts of mouse tissues were prepared by homogenization in 10 volumes of potassium phosphate buffer, pH 6.5, using an Ultra-turrax and centrifugation for 20 min at 15,000 × g. The mouse intestine extract was adjusted to pH 5.0 by the addition of citric acid (0.2 m); NaCl was added to a final concentration of 2m. A chitin column was prepared by mixing 10 g of swollen Sepharose G25 fine (Amersham Pharmacia Biotech) with 300 mg of colloidal chitin, followed by equilibration with phosphate-buffered saline containing 2 m NaCl. The extracts were applied onto the column with a flow speed of 0.4 ml/min. After extensive washing, bound chitinase was eluted from the column with 8 m urea, which was subsequently removed by dialysis. Protein concentrations were determined according to the method of Lowry et al. (17Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as a standard. Fractions containing chitinase activity were subjected to SDS-PAGE and Western blotting as described (8Renkema G.H. Boot R.G. Muijsers A.O. Donker-Koopman W.E. Aerts J.M.F.G. J. Biol. Chem. 1995; 270: 2198-2202Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). N-terminal protein sequencing was performed as described using a Procise 494 sequencer (Applied Biosystems Perkin Elmer) (8Renkema G.H. Boot R.G. Muijsers A.O. Donker-Koopman W.E. Aerts J.M.F.G. J. Biol. Chem. 1995; 270: 2198-2202Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Colloidal chitin was prepared as described by Shimahara and Takiguchi (18Shimahara K. Takiguchi Y. Methods Enzymol. 1988; 166: 417-423Crossref Scopus (179) Google Scholar). SDS-PAGE was performed with a Amersham Pharmacia Biotech phast gel system, according to the instructions of the manufacturer, using 12.5‥ polyacrylamide gels, followed by silver staining. Glycol-chitin electrophoresis was conducted as described by Escott and Adams (19Escott G.M. Adams D.J. Infect. Immun. 1995; 63: 4770-4773Crossref PubMed Google Scholar), except for an extension of the renaturation time to 8 h. Glycol-chitin was prepared from glycol chitosan (Sigma) as described by Trudel and Asselin (20Trudel J. Asselin A. Anal. Biochem. 1989; 178: 362-366Crossref PubMed Scopus (479) Google Scholar). The native isoelectric point of chitinases was determined by flat bed isoelectric focusing in granulated Ultrodex gels (Amersham Pharmacia Biotech) as described (8Renkema G.H. Boot R.G. Muijsers A.O. Donker-Koopman W.E. Aerts J.M.F.G. J. Biol. Chem. 1995; 270: 2198-2202Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Total RNA was isolated using RNAzol B (Biosolve, Barneveld, The Netherlands) according to the instructions of the manufacturer. Northern blots, using 15 μg of total RNA, were performed as described (9Boot R.G. Renkema G.H. Strijland A. van Zonneveld A.J. Aerts J.M.F.G. J. Biol. Chem. 1995; 270: 26252-26256Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Human and mouse RNA Master Blots (CLONTECH, Palo Alto, CA) were used to examine the tissue distribution of transcripts according to the instructions of the manufacturer. The following probes were used: the full-length mouse acidic chitinase cDNA, the human EST clone oq35c04.s1 (GenBankTM accession number AA976830) and glyceraldehyde-3-phosphate dehydrogenase as control. Radiolabeling and hybridization was conducted as described previously (9Boot R.G. Renkema G.H. Strijland A. van Zonneveld A.J. Aerts J.M.F.G. J. Biol. Chem. 1995; 270: 26252-26256Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Quantification of radioactivity was performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Reverse transcription polymerase chain reaction (PCR) fragments were generated from mouse lung total RNA using degenerate oligonucleotides, as described (9Boot R.G. Renkema G.H. Strijland A. van Zonneveld A.J. Aerts J.M.F.G. J. Biol. Chem. 1995; 270: 26252-26256Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Obtained fragments were cloned in pGEM-T (Promega, Madison, WI), sequenced, and compared with the amino acid sequence established by N-terminal protein sequencing. A comparison with the GenBankTM mouse EST (expressed sequence tag) data base using the Basic local alignment search tool (BLAST) at the National Center for Biotechnology Information showed that several EST clones matched the mouse chitinase cDNA sequence, for example, ms33 h09.y1 (GenBankTM accession number AI892792). This clone was obtained and sequenced. Antisense primers were generated complementary to the most 3′ region of the EST clone (A tail primer, 5′-TTTTGGCTACCAATTTTATTGC-3′) and two internal antisense primers (MAS1, 5′-CAGCTACAGCAGCAGTAACCATC-3′ and MAS2, 5′-TTCAGGGATCTCATAGCCAGC-3′). The MAS1 and MAS2 primers were used to clone the most 5′ end of the mouse acidic chitinase cDNA using 5′ rapid amplification of cDNA ends and the Marathon-Ready mouse Lung cDNA kit (CLONTECH) according to the instructions of the manufacturer. To obtain the complete coding sequence a 5′ sense primer was generated (MS1, 5′-CGATGGCCAAGCTACTTCTCGT-3′). The total cDNA sequence was subsequently generated using MS1 and the A tail primer. The fragments of two independent PCRs were cloned into pGEM-T (Promega), and the nucleotide sequences of two independent clones from each PCR were sequenced from both strands by the procedure of Sanger using fluorescent nucleotides on an Applied Biosystems 377A automated DNA sequencer following Applied Biosystems protocols. Comparison of the mouse AMCase cDNA sequence with the human EST data base (National Center for Biotechnology Information) revealed the presence of a human EST clone oq35c04.s1 (GenBankTM accession number AA976830) highly homologous to the mouse acidic chitinase. Following the same strategy, the full-length human AMCase cDNA was cloned using human stomach total RNA (CLONTECH) for the reverse transcription PCR with the same degenerate primers. A human Marathon-Ready Lung cDNA was used to clone the most 5′ end of the cDNA by 5′ rapid amplification of cDNA ends using the following primers: HAS2 (5′-TCTGACAGCACAGAATCCACTGCC-3′) and HAS3-A tail (5′-TTGACTGCTGATTTTATTGCAG-3′). The total cDNA sequence was subsequently generated using HS1 (5′-GCTTTCCAGTCTGGTGGTGAAT-3′) and HAS3-A tail. The fragments of two independent PCRs were cloned in pGEM-T (Promega) and sequenced as described above. Transient expression of the various cDNAs in COS-1 cells was performed exactly as described previously (9Boot R.G. Renkema G.H. Strijland A. van Zonneveld A.J. Aerts J.M.F.G. J. Biol. Chem. 1995; 270: 26252-26256Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). To obtain more insight into the potential occurrence of multiple mammalian chitinases, tissues of mouse and rat were examined for chitinolyic activity using the chitin-like 4-methylumbelliferyl-β-chito-oligosaccharide substrates. In extracts of stomach and intestine, a high level of activity was detected, whereas extracts of lung, tongue, kidney, and plasma showed significant but lower activities. Isoelectric focusing of a mouse lung extract revealed a major peak of chitinolytic activity with pI of 4.5, whereas minor peaks were found with pI levels of 5.5–6.5 (Fig.1). Extracts of other mouse and rat tissues showed similar profiles of chitinolytic activity upon isoelectric focusing. The observed rodent chitinase with acidic isoelectric point (pI 4.5 form) differs strikingly from human chitotriosidase which has an apparent neutral/basic pI. The mouse acidic chitinase activity was found to bind to chitin particles with high affinity. Chitin affinity chromatography was used to purify the enzyme, as described under “Experimental Procedures.” The procedure resulted in a 30,000-fold purification of an apparently homogeneous 50-kDa protein. The specific activity of the purified enzyme was 3.9 nmol of 4-methylumbelliferyl-chitotrioside hydrolyzed per mg per hour at pH 5.2, which is almost identical to that of human chitotriosidase. The N-terminal amino acid sequence of purified acidic chitinase was determined (Fig. 2) and was found to be almost identical to that of other known members of the chitinase family. This amino acid sequence allowed the cloning of the corresponding full-length mouse acidic chitinase cDNA, as described under “Experimental Procedures.” The full-length cDNA predicts the synthesis of a 50-kDa (pI 4.85) protein with a characteristic signal peptide (Fig. 2). Expression of this cDNA in COS-1 cells led to the secretion of an 50-kDa active chitinase with a pI of 4.8. The mouse acidic chitinase protein shows considerable sequence homology to human chitotriosidase. Comparison of the amino acid sequence of both mature proteins revealed an identity of 52‥ and a similarity of 60‥. Like the human chitotriosidase, the mouse enzyme is predicted to contain an N-terminal catalytic domain of about 39 kDa, a hinge region, and a C-terminal chitin binding domain (Fig. 2). The mouse acidic chitinase, like chitotriosidase, is predicted to lackN-linked oligosaccharides, explaining the observed absence of binding to concanavalin A (data not shown). Several different assays revealed that the mouse acidic chitinase is able to degrade chitin and therefore has to be considered to be a true chitinase. Firstly, fluorophore-assisted carbohydrate electrophoresis analysis revealed that recombinant mouse chitinase, like chitotriosidase, releases mainly chitobioside fragments from chitin (Fig. 3). Secondly, like chitotriosidase and some other nonmammalian chitinases, the mouse acidic chitinase is strongly inhibited (IC50 of 0.4 μm) by the competitive chitinase inhibitor allosamidin (21Milewski S. O'Donnel R.W. Gooday G.W. J. Gen. Microbiol. 1992; 138: 2545-2550Crossref PubMed Scopus (28) Google Scholar, 22Dickinson K. Keer V. Hitchcock C.A. Adams D.J. J. Gen. Microbiol. 1989; 135: 1417-1421PubMed Google Scholar, 23McNab R. Glover L.A. FEMS Microbiol. Lett. 1991; 82: 79-82Crossref Scopus (19) Google Scholar). Finally, the mouse acidic chitinase and chitotriosidase were both able to digest chitin in the cell wall of regenerating spheroplasts of C. albicans. The chitin content of the cell wall was determined with the Calcofluor white stain (see “Experimental Procedures”). When regenerating cells were incubated for 2 h with 3 μg/ml recombinant chitotriosidase or 3 μg/ml recombinant mouse acidic chitinase, the chitin content was reduced by 27 and 33‥, respectively. Concomitant presence of allosamidin during the incubation completely abolished the effect of both recombinant chitinases. The apparent molecular masses of identically produced recombinant human chitotriosidase and recombinant mouse acidic chitinase are comparable when run on a SDS-PAGE gel under reducing conditions. However, under nonreducing conditions, the mouse acidic chitinase migrates significantly slower than the human chitotriosidase (Fig.4 A). Upon gelelectrophoresis (under nonreducing conditions) in polyacrylamide gels containing glycolchitin, followed by regeneration of active enzyme and detection of the local digestion of glycolchitin using Calcofluor staining, the mouse acidic chitinase migrates slightly faster than human chitotriosidase (Fig. 4 B). A further striking difference between human chitotriosidase and the mouse acidic chitinase is their behavior at acidic pH. The mouse acidic chitinase shows a pronounced pH optimum at pH 2.3 and a less pronounced optimum at more neutral pH (pH 4–7). Chitotriosidase, however, shows only a broad pH optimum (Fig.5 A) and is completely inactivated by pre-incubation at low pH (Fig. 5 B). In the presence of 0.5‥ (w/v) trichloroacetic acid 58‥ of chitotriosidase is precipitated, whereas under similar circumstances the mouse acidic chitinase remains in solution. At 2.5‥ (w/v) trichloroacetic acid all chitotriosidase precipitates, whereas 26‥ of mouse acidic chitinase remains unprecipitated (Fig. 5 C). Another major difference between human chitotriosidase and the mouse acidic chitinase is revealed by comparison of RNA expression patterns. Although human chitotriosidase mRNA is mainly found in lymph node, bone marrow, and lung, the mouse acidic chitinase mRNA is predominantly found in stomach, submaxillary gland, and, at a lower level, in the lung (Fig. 6). Surprisingly, no mouse acidic chitinase mRNA can be detected in the small intestine, suggesting that the protein in the intestine is probably derived from the upper parts of the gastrointestinal tract, such as the stomach. In rat tissues a comparable acidic chitinase was observed. Our findings indicate that the acidic chitinase in rodents is distinct from human chitotriosidase. The discrete enzyme is therefore referred to as acidic mammalian chitinase or AMCase. It was investigated whether such an acidic chitinase is also present in man. Screening the human EST data base at the National Center for Biotechnology Information with the mouse acidic chitinase cDNA revealed the presence of a highly homologous human EST clone (oq35c04.s1, GenBankTM accession number AA976830). The tissue distribution of this human mRNA was examined using a human Masterblot (CLONTECH). The expression pattern of this mRNA is similar to the expression pattern of the mouse acidic chitinase (Fig.7), being highly expressed in the stomach and at a lower level in the lung. Using degenerate oligonucleotides directed against members of the chitinase family, we were able to amplify other regions of the human acidic chitinase, generating enough information to clone the full-length human acidic chitinase cDNA (Fig. 8 A). Screening the GenBankTM data base using the full-length human cDNA revealed that it was almost identical to TSA1902-L (GenBankTM accession number AB025008) and TSA1902-S (GenBankTM accession number AB025009) from a lung cDNA library described by Saito et al. (24Saito A. Ozaki K. Fujiwara T. Nakamura Y. Tanigami A. Gene (Amst.). 1999; 239: 325-331Crossref PubMed Scopus (28) Google Scholar). These two sequences are most probably splice variants of the human acidic chitinase mRNA. Only expression of full-length human AMCase cDNA in COS-1 cells led to the production of a protein with chitinolytic activity (data not shown). Sequence comparison of the human acidic chitinase and the mouse acidic chitinase revealed an 82‥ identity and a similarity of 86‥ (Fig. 8 B).Figure 8Human AMCase cDNA sequence and deduced amino acid sequence. A, the human AMCase cDNA sequence (GenBankTM accession number AF290004) is indicated by the upper sequence, and the deduced amino acid sequence is indicated below the nucleotide sequence. The characteristic hydrophobic signal peptide (amino acids 1–21) isunderlined with a single line. B, amino acid sequence comparison of mature (without signal peptide) human (h) and mouse (m) AMCase and human chitotriosidase. Residues conserved among at least two out of the three sequences are boxed.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The demonstration by Saito et al. (24Saito A. Ozaki K. Fujiwara T. Nakamura Y. Tanigami A. Gene (Amst.). 1999; 239: 325-331Crossref PubMed Scopus (28) Google Scholar) that the gene encoding TSA1902 is located on chromosome 1p13 indicates that mammals contain indeed at least two discrete genes that encode functional chitinases, being chitotriosidase (locus 1q32) and AMCase (locus 1p13). Definitive proof for the existence of at least two distinct, functional mammalian chitinase genes was recently obtained by the partial cloning of chitotriosidase cDNA from the rat. The cloned rat cDNA (80‥ of the complete cDNA) encodes a protein that is 80‥ identical to the human counterpart. For many years the existence of chitinase has been well documented for a large variety of organisms, including bacteria, plants, insects, and fungi (for an overview see Ref. 6Flach J. Pilet P.E. Jolles P. Experientia. 1992; 48: 701-716Crossref PubMed Scopus (332) Google Scholar). More recently, it has become clear that mammals also contain such enzymes. 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