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Transglycosidase Activity of Chitotriosidase

2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês

10.1074/jbc.m301804200

1083-351X

Begoña Aguilera, Karen Ghauharali‐van der Vlugt, Mariette T.J. Helmond, Jos M.M. Out, Wilma E. Donker‐Koopman, J.E.M. Groener, Rolf G. Boot, G. Herma Renkema, Gijs A. van der Marel, Jacques H. van Boom, Herman S. Overkleeft, Johannes M. F. G. Aerts,

Trypanosoma species research and implications

Chitotriosidase is a chitinase that is massively expressed by lipid-laden tissue macrophages in man. Its enzymatic activity is markedly elevated in serum of patients suffering from lysosomal lipid storage disorders, sarcoidosis, thalassemia, and visceral Leishmaniasis. Monitoring of serum chitotriosidase activity in Gaucher disease patients during progression and therapeutic correction of their disease is useful to obtain insight in changes in body burden on pathological macrophages. However, accurate quantification of chitotriosidase levels by enzyme assay is complicated by apparent substrate inhibition, which prohibits the use of saturating substrate concentrations. We have therefore studied the catalytic features of chitotriosidase in more detail. It is demonstrated that the inhibition of enzyme activity at excess substrate concentration can be fully explained by transglycosylation of substrate molecules. The potential physiological consequences of the ability of chitotriosidase to hydrolyze as well as transglycosylate are discussed. The novel insight in transglycosidase activity of chitotriosidase has led to the design of a new substrate molecule, 4-methylumbelliferyl-(4-deoxy)chitobiose. With this substrate, which is no acceptor for transglycosylation, chitotriosidase shows normal Michaelis-Menten kinetics, resulting in major improvements in sensitivity and reproducibility of enzymatic activity measurements. The novel convenient chitotriosidase enzyme assay should facilitate the accurate monitoring of Gaucher disease patients receiving costly enzyme replacement therapy. Chitotriosidase is a chitinase that is massively expressed by lipid-laden tissue macrophages in man. Its enzymatic activity is markedly elevated in serum of patients suffering from lysosomal lipid storage disorders, sarcoidosis, thalassemia, and visceral Leishmaniasis. Monitoring of serum chitotriosidase activity in Gaucher disease patients during progression and therapeutic correction of their disease is useful to obtain insight in changes in body burden on pathological macrophages. However, accurate quantification of chitotriosidase levels by enzyme assay is complicated by apparent substrate inhibition, which prohibits the use of saturating substrate concentrations. We have therefore studied the catalytic features of chitotriosidase in more detail. It is demonstrated that the inhibition of enzyme activity at excess substrate concentration can be fully explained by transglycosylation of substrate molecules. The potential physiological consequences of the ability of chitotriosidase to hydrolyze as well as transglycosylate are discussed. The novel insight in transglycosidase activity of chitotriosidase has led to the design of a new substrate molecule, 4-methylumbelliferyl-(4-deoxy)chitobiose. With this substrate, which is no acceptor for transglycosylation, chitotriosidase shows normal Michaelis-Menten kinetics, resulting in major improvements in sensitivity and reproducibility of enzymatic activity measurements. The novel convenient chitotriosidase enzyme assay should facilitate the accurate monitoring of Gaucher disease patients receiving costly enzyme replacement therapy. Chitin, the linear polymer of β 1,4-linked N-acetylglucosamine, is a structural component of cell walls and coatings of many organisms. For a long time it was thought that mammals are unable to produce endoglucosaminidases that fragment chitin. Investigations on Gaucher disease led to the serendipitous discovery of a functional endogenous chitinase in man (1Hollak 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 (760) Google Scholar, 2Renkema 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 (269) Google Scholar, 3Boot 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 (339) Google Scholar). It was observed that serum samples from Gaucher patients show a 1000-fold elevated capacity to hydrolyze 4-methylumbelliferyl-β-d-chitotriose. The responsible enzyme, named chitotriosidase, was shown to be able to cleave natural chitin and a variety of artificial chitin-like substrates such as 4-methylumbelliferyl and p-nitrophenyl chitooligosaccharides. Chitotriosidase belongs to the family 18 of glycosylhydrolases and is highly homologous to chitinases from lower organisms. The enzyme consists of a N-terminal TIM-barrel structure containing the catalytic groove and a C-terminal chitin-binding domain connected by a short hinge region. The crystal structure of the 39-kDa catalytic domain and its complexes with a chitooligosaccharide and allosamidin has been described recently (4Fusetti F. von Moeller H. Houston D. Rozeboom H.J. Dijkstra B.W. Boot R.G. Aerts J.M. van Aalten D.M. J. Biol. Chem. 2002; 277: 25537-25544Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). The structures reveal an elongated active site cleft, compatible with binding long chitin polymers. The catalytic center of chitotriosidase closely resembles that of chitinases from lower organisms like chitinase A/B from Serratia marcescens. The catalytic reaction of family chitinases takes place through a retaining mechanism, resulting in β-anomers by hydrolysis of β 1,4-glycosidic linkages. The reaction is initiated by distortion of the pyranose ring of the (–1) sugar moiety in the active site and protonation of the glycosidic oxygen by a protonated acidic residue forming a covalent oxazolinium ion intermediate. The subsequent nucleophilic attack differs from classical reaction mechanisms of retaining enzymes such as lysozyme (5Davies G.J. Mackenzie L. Varrot A. Dauter M. Brzozowski A.M. Schulein M. Withers S.G. Biochemistry. 1998; 37: 11707-11713Crossref PubMed Scopus (238) Google Scholar) in that it involves the N-acetyl group of the (–1) sugar, rather than a carboxylate side chain on the protein, a so-called substrate-assisted mechanism (6Terwisscha van Scheltinga A.C. Armand S. Kalk K.H. Isogai A. Henrissat B. Dijkstra B.W. Biochemistry. 1995; 34: 15619-15623Crossref PubMed Scopus (309) Google Scholar, 7van Aalten D.M. Komander D. Synstad B. Gaseidnes S. Peter M.G. Eijsink V.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8979-8984Crossref PubMed Scopus (395) Google Scholar). Chitotriosidase is selectively expressed in chronically activated tissue macrophages, like the lipid-laden storage cells that accumulate in large quantities in various tissues of Gaucher patients (8Aerts J.M. Hollak C.E. Baillieres Clin. Haematol. 1997; 10: 691-709Abstract Full Text PDF PubMed Scopus (140) Google Scholar). Tissue macrophages largely secrete newly synthesized 50-kDa chitotriosidase, but about one-third is directly routed to lysosomes and proteolytically processed to the 39-kDa unit that remains catalytically active (9Renkema 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). Intriguingly, one in every three individuals from various ethnic groups carries one abnormal chitotriosidase gene with a 24-bp duplication that prevents production of enzyme (10Boot 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 (345) Google Scholar). About 6% of the population is homozygous for this mutant allele and consequently completely lacks chitotriosidase activity. In analogy to the function of homologous chitinases in plants, the physiological role of chitotriosidase is most likely in innate immunity toward chitin containing pathogens. An increased risk for nematode infection has indeed been described for chitotriosidase-deficient individuals (11Choi E.H. Zimmerman P.A. Foster C.B. Zhu S. Kumaraswami V. Nutman T.B. Chanock S.J. Genes Immun. 2001; 2: 248-253Crossref PubMed Scopus (125) Google Scholar). Pathological tissue macrophages in specific disease conditions massively express chitotriosidase. A shared feature of such cells is accumulation of lipid material in the lysosomal apparatus. The molecular mechanism that underlies the tightly controlled expression of chitotriosidase is not yet known. In serum of patients suffering from lysosomal lipid storage disorders (12Guo Y. He W. Boer A.M. Wevers R.A. de Bruijn A.M. Groener J.E. Hollak C.E. Aerts J.M. Galjaard H. van Diggelen O.P. J. Inherit. Metab. Dis. 1995; 18: 717-722Crossref PubMed Scopus (185) Google Scholar), thalassemia (13Barone R. Di Gregorio F. Romeo M.A. Schiliro G. Pavone L. Blood Cells Mol. Dis. 1999; 25: 1-8Crossref PubMed Scopus (68) Google Scholar), sarcoidosis, and visceral Leishmaniasis (1Hollak 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 (760) Google Scholar), as well as in cerebral spine fluid of patients with multiple sclerosis, significantly elevated chitotriosidase activities are detectable using artificial chromogenic and fluorogenic substrates. The abnormality is exploited for diagnostic and monitoring purposes. For example, the efficacy of the extremely costly enzyme replacement therapy of Gaucher patients is monitored by following corrections in plasma chitotriosidase activity levels (8Aerts J.M. Hollak C.E. Baillieres Clin. Haematol. 1997; 10: 691-709Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 14Mistry P.K. Abrahamov A. Baillieres Clin. Haematol. 1997; 10: 817-838Abstract Full Text PDF PubMed Scopus (31) Google Scholar). However, accurate quantification of chitotriosidase levels by enzyme assay is prohibited by apparent substrate inhibition (1Hollak 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 (760) Google Scholar). The inevitable use of subsaturating substrate concentrations reduces the reproducibility and sensitivity of the assay and has hampered its wide-scale application outside expert laboratories. We have studied the molecular basis for the apparent substrate inhibition of chitotriosidase in more detail. The information obtained allowed us to design a novel artificial substrate that can be used for a substantially improved enzyme activity assay. The results of the study are here reported and discussed. Human Recombinant 50-kDa Chitotriosidase—Enzyme was purified from the medium of stably transfected baby hamster kidneys cells using the full-length human chitotriosidase cDNA cloned in the pNUT vector (4Fusetti F. von Moeller H. Houston D. Rozeboom H.J. Dijkstra B.W. Boot R.G. Aerts J.M. van Aalten D.M. J. Biol. Chem. 2002; 277: 25537-25544Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). The enzyme was purified by sequential chromatofocussing, isoelectrofocussing, and gel filtration (2Renkema 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 (269) Google Scholar). Chitin oligosaccharides were purchased from Seikagu Kogyo. All other chemicals, including 4-methylumbelliferone, p-nitrophenol, and their conjugates with chitooligosaccharides, were purchased from Sigma, Aldrich, or Baker. Enzyme Activity Measurements—The conventional enzyme assays with commercial artificial fluorogenic or chromogenic substrates were performed exactly as described before (1Hollak 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 (760) Google Scholar). Briefly, enzyme preparations were incubated at 37 °C with 22 μm 4-MU 1The abbreviations used are: 4-MU, 4-methylumbelliferyl; PNP, p-nitrophenyl; HPLC, high performance liquid chromatograpy; AMCase, acidic mammalian chitinase.-chitooligosaccharides or with 220 μm PNP-chitooligosaccharides in McIlvain buffer (100 mm citric acid, 200 mm sodium phosphate, pH 5.6) containing 1 mg/ml bovine serum albumin. The enzyme assay was stopped by addition of excess 0.3 m glycine-NaOH, pH 10.3. Formed 4-methylumbelliferone was detected fluorometrically (excitation at 445 nm; emission at 366 nm); formed PNP was detected spectrophotometrically at 405 nm. The optimal assay conditions with the newly synthesized 4-MU-(4-deoxy)chitobiose were at 114 μm in the same buffer. A 35 mm stock solution of the novel substrate was prepared in Me2SO. Chitooligosaccharide Analysis—Isocratic HPLC chromatography with a Prevail carbohydrate ES column (Alltech), and UV detection at 214 nm was used to analyze chitooligosaccharides. The injection volume was 10 μl, the flow rate of the eluent (62:28 (w/w) acetonitrile:H2O) was 1 ml/min. Synthesis of the Novel Substrate 4-MU-(4-deoxy)chitobiose—Octaacetyl chitobioside (1), prepared by acetolysis of chitin (15Inaba T. Ohgushi T. Iga T. Hasegawa E. Chem. Pharm. Bull. 1984; 32: 1597-1603Crossref Scopus (23) Google Scholar), was transformed into the partially protected disaccharide (2) according to a previously reported procedure (16Shaban M. Jeanloz R.W. Carbohydr. Res. 1971; 19: 311-318Crossref PubMed Scopus (28) Google Scholar). Hydrolysis of the acetate groups followed by regioselective introduction of the 4″-6″-benzylidene group, acetylation and final acidic removal of the benzylidene moiety, rendered 2 with a yield of 41%. Regioselective benzoylation of the 6″-hydroxyl function in 2 with 1-(benzyloxy)benzotriazole (17Kim S. Chang H. Kim W.J. J. Org. Chem. 1985; 50: 1751-1752Crossref Scopus (87) Google Scholar) and subsequent esterification with 1,1′-thiocarbonyldiimidazole rendered key intermediate 3 with a 53% yield. Barton deoxygenation (18Barton D.H.R. McCombie S.W. J. Chem. Soc. Perkin Trans. I. 1975; : 1574-1585Crossref Scopus (1973) Google Scholar) of 3 was accomplished by treatment with tributyltin hydride and a catalytic amount of azoisobutyronitrile in toluene, yielding 4″-deoxyheptaacetyl chitobioside (4) with a 72% yield. Subsequent chlorination to 5, followed by condensation with sodium 4-methylumbelliferonate, and final de-O-esterification (19Delmotte F.M. Privat J.-P.D.J. Monsigny M.L. Carbohydr. Res. 1975; 40: 353-364Crossref Scopus (41) Google Scholar) rendered homogeneous 4-MU-(4″-deoxy)chitobiose, compound 6, with a 20% yield. Transglycosylation—The complex relationship of increasing concentrations of 4-methylumbelliferyl chitobiose, triose, and tetraose substrates with the rate of fluorescent (4-MU; 4-methylumbelliferone) product formation is depicted for pure recombinant chitotriosidase (Fig. 1). It can be seen that at higher substrate concentrations less product is formed, nearly independent of the chemical nature of the substrate. Identically shaped curves were obtained for the corresponding p-nitrophenyl substrates and the rate of colored p-nitrophenol formation (not shown). This suggests that the concentration of N-acetylglucosamine moieties at the non-reducing end of the substrate molecules is somehow critical. The finding prompted us to examine the possibility of some transglycosylation event involving these moieties. For this purpose, pure recombinant chitotriosidase was incubated with 4-methylumbelliferyl-β-N-acetylglucosamine, p-nitrophenyl chitobiose, or a combination of both compounds. Fig. 2 shows that, as expected, no fluorescent 4-MU is formed upon incubation of enzyme with both compounds separately. In sharp contrast, incubation with the mixture of both compounds results in formation of fluorescent 4-MU after a lag phase. Transglycosylation offers an attractive explanation for the outcome of the experiment, as schematically presented in Fig. 2 (right side). As an initial step, chitotriosidase removes a chitobiose moiety from the PNP-chitobiose substrate. Next, the oxazolinium transition state intermediate may either use H2O or the hydroxyl at the C-4 position in 4-MU-N-acetylglucosamine as acceptor. The latter reaction results in formation of 4-MU-chitotriose, which after some time is cleaved by chitotriosidase to yield fluorescent 4-MU.Fig. 2Demonstration of transglycosylation with artificial chitooligosaccharides. Left-hand side, recombinant 50-kDa chitotriosidase was incubated for different time periods with 2.7 mm PNP-chitobiose (A), with 1.6 mm 4-MU-N-acetylglucosamine (B), or with the mixture of both substrates (C). Released 4-MU (▪) and PNP (▴) were detected as described under "Materials and Methods." arbs, arbitrary units. Right-hand side, explanation of result.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate the ability of chitotriosidase to transglycosylate natural chitooligosaccharides, pure recombinant enzyme was incubated with 4-methylumbelliferyl-β-N-acetylglucosamine and chitopentaose of p-nitrophenyl chitobiose. 10-fold larger amounts of fluorescent 4-MU are formed in the presence of chitopentaose as compared with p-nitrophenyl chitobiose, indicating that the natural oligosaccharide can act as donor in the transglycosylation reaction. Fig. 3 shows that upon incubation of 5 mm chitopentaose with recombinant chitotriosidase, not only chitobiose and chitotriose, but also chitotetraose, and even chitohexaose and chitoheptaose, are formed. Since chitotriosidase is unable to remove or add a single N-acetylglucosamine moiety, formation of chitotetraose demonstrates that chitotriosidase can exert transglycosidase activity toward chitooligosaccharides. The formation of small amounts of chitoheptaose points in the same direction. The total recovery of glucosamine units in the experiment is virtually 100%. It was noticed that meanwhile unequal amounts of chitobiose and chitotriose are formed. Apparently, the transglycosidase activity of chitotriosidase is very considerable, and predominantly chitobiose is donated to accepting chitooligosaccharides. The apparent substrate inhibition with 4-MU-chitooligosaccharide substrates can be fully explained by the transglycosidase capacity of chitotriosidase (see Scheme 1). With increasing concentration of 4-MU-chitobiose there is increasing formation of 4-MU-chitotetraose by transglycosidase activity. Chitobiose units can be subsequently transferred from 4-MU-chitotetraose to 4-MU-chitobiose molecules, a futile cycle generating no new products and not releasing fluorescent 4-MU. The experimenter observes this process as inhibition of enzyme activity, since that is monitored by formation of fluorescent 4-MU. Design and Synthesis of Novel Substrate—The realization that ongoing transglycosylation is the cause of the problems encountered with the measurement of enzymatic activity of chitotriosidase using artificial chitooligosaccharide substrates stimulated us to design a novel substrate molecule that is lacking a hydroxyl at the C-4 position of the non-reducing N-acetylglucosamine. A novel compound, 4-methylumbelliferyl-N-acetylglucosamine-4,1-(4-deoxy)-N-acetylglucosamine (4-MU-(4-deoxy)chitobiose) was synthesized, exactly as described under "Materials and Methods" (see Fig. 4 for its chemical structure and route of synthesis). We envisioned that this compound should only be able to act as substrate for hydrolysis but not as acceptor for transglycosylation. Fig. 5 shows for pure recombinant chitotriosidase the relationship between the rate of fluorescent 4-MU formation and the concentration of 4-MU-chitobiose and 4-MU-(4-deoxy)chitobiose, respectively. Normal Michaelis-Menten kinetics occurs with 4-MU-deoxychitobiose as substrate, sharply contrasting to the marked substrate inhibition with 4-MU-chitobiose. This finding is in accordance with the fact that the novel substrate 4-MU-deoxychitobiose cannot undergo transglycosylation.Fig. 5Chitotriosidase activity toward 4-MU-chiobiose and its deoxy variant. Human recombinant chitotriosidase was incubated for 15 min with increasing concentrations of 4-MU substrates, and released 4-MU was detected fluorometrically as described under "Materials and Methods." Activity toward 4-MU-chitobiose (▪), activity toward 4-MU-deoxy-chiotbiose (▴) expressed in arbitrary units (Arbs).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Application of Novel Chitotriosidase Assay—The optimal enzyme activity assay conditions were determined for the novel deoxy-substrate. Maximal activity of recombinant chitotriosidase toward the substrate occurs at pH 5.6 with an apparent Km of about 50 micromolar. An assay mixture composed of 114 μm of the deoxy-substrate in 0.1/0.2 m McIlvaine buffer, pH 5.6, allows the measurement of enzyme activity at maximal rate. Production of 4-MU is directly proportional to enzyme input and linear in time over a broad range. The standard assay mixture described above also allows reliable detection of the endogenous chitotriosidase in biological samples like plasma, serum, cerebral spine fluid, urine and extracts of blood cells, cultured macrophages, and tissues (not shown). This was further substantiated by the spiking of increasing amounts of recombinant chitotriosidase in the various biological samples. Linearity with time and proportionality to sample input were also examined. For reason of maximal sensitivity, subsaturating concentrations of 4-MU-chitobiose or 4-MU-chitotriose had to be used in enzymatic assays (1Hollak 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 (760) Google Scholar). Although the intra-assay reproducibility with the published method is satisfactory, the inter-assay reproducibility is intrinsically poor due to unavoidable differences in precise substrate concentration. Moreover, the proportionality with enzyme input of such assays is relatively limited. In comparison with enzyme assays with the conventional 4-MU-chitobiose or 4-MU-chitotriose substrate, the enzyme assay with the novel deoxy-substrate is far more sensitive, linear in time, and directly proportional to enzyme input over a far larger range (see Fig. 6 for an illustration). Nowadays plasma chitotriosidase activity levels are determined to assist in the clinical management of Gaucher patients. Plasma chitotriosidase has evolved as an important tool in decision making in the clinic. For example, initiation of costly enzyme replacement therapy for Gaucher patients is considered in many centers when the plasma chitotriosidase level exceeds a critical threshold level and changes in activity level serve as guideline for optimalization of dosing regimens during enzyme replacement therapy of Gaucher patients (8Aerts J.M. Hollak C.E. Baillieres Clin. Haematol. 1997; 10: 691-709Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 14Mistry P.K. Abrahamov A. Baillieres Clin. Haematol. 1997; 10: 817-838Abstract Full Text PDF PubMed Scopus (31) Google Scholar). Obviously, the application of plasma chitotriosidase measurements for these purposes requires highly reliable and reproducible data while monitoring patients over a large period of time. The intrinsic limitation of the current chitotriosidase activity measurement with non-constant and non-saturating concentrations of 4-MU-chitobiose or 4-MU-chitotriose as substrates is the difficulty to ensure that the measured reaction rate is reproducibly proportional to enzyme concentration (1Hollak 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 (760) Google Scholar). In our experience, the only way this can be (nearly) accomplished when using the conventional 4-MU-chitooligosaccharide substrates is to vary enzyme input meticulously to obtain a fixed amount of product formation in a defined assay time period. This laborious approach implies that a sample has to be measured in duplicate at various dilutions. In sharp contrast, the assay with the novel substrate allows measurement at maximal reaction rate that is continuously proportional to enzyme concentration. The novel assay is therefore far more robust and does not require the tedious precautions associated with the current assay. A significant additional advantage of the assay with the novel substrate is the ∼5-fold increase in sensitivity of detection. This is extremely helpful for diagnostic applications in relation to a variety of disease conditions with relatively modest (2–10-fold) plasma chitotriosidase elevations like sarcoidosis, thalassemia, and lysosomal lipid storage disorders. Our demonstration of a marked transglycosidase capacity of chitotriosidase is not entirely unexpected based on some very recent literature reports. During the course of our investigation it was described that family 18 chitinases from plants and microbes are to a variable extent able to catalyze transglycosylation reactions. A comparative study revealed that plant family 18 chitinases have a (–4)(–3)(–2)(–1)(+1)(+2)-type binding cleft, while the microbial family 18 chitinases show a (–2)(–1)(+1)(+2)(+3)(+4)-type binding cleft. Transglycosylation is found in the latter, but not in the former. Soaking of human chitotriosidase crystals with chitooligosaccharides has so far not been informative in this respect. Despite carrying out the experiments at pH 10.6 hydrolysis occurred, rendering electron density maps where only an ordered N-acetylglucosamine dimer could be observed at subsites –2 and –1 (4Fusetti F. von Moeller H. Houston D. Rozeboom H.J. Dijkstra B.W. Boot R.G. Aerts J.M. van Aalten D.M. J. Biol. Chem. 2002; 277: 25537-25544Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). The production of recombinant human chitotriosidase with one key active site residue, Glu-140, mutated to Asp or Leu has been earlier described by us (20Renkema G.H. Boot R.G. Au F.L. Donker-Koopman W.E. Strijland A. Muijsers A.O. Hrebicek M. Aerts J.M. Eur. J. Biochem. 1998; 251: 504-509Crossref PubMed Scopus (334) Google Scholar). The inactive mutant protein has retained its ability to bind chitooligosaccharides in the catalytic cleft. The soaking of crystals of inactive mutant chitotriosidase with chitooligosaccharides should render a better insight in reaction mechanism and structural factors controlling enzyme function. It will be of interest to study in greater detail the prerequisites in the catalytic center of chitotriosidase to act as a transglycosidase. Informative should also be a comparison of chitotriosidase with a highly related, human gastrointestinal chitinase (21Boot R.G. Blommaart E.F. Swart E. Ghauharali-van der Vlugt K. Bijl N. Moe C. Place A. Aerts J.M. J. Biol. Chem. 2001; 276: 6770-6778Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). The enzyme was very recently identified and characterized by us and named acidic mammalian chitinase (AMCase) (21Boot R.G. Blommaart E.F. Swart E. Ghauharali-van der Vlugt K. Bijl N. Moe C. Place A. Aerts J.M. J. Biol. Chem. 2001; 276: 6770-6778Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). Despite the high degree of homology with the macrophage chitotriosidase, AMCase has a number of distinct features. Interestingly, AMCase uniquely shows a dual pH optimum for hydrolysis. A second optimum of hydrolytic activity occurs at very low pH (about pH 2.0). AMCase shows a different ratio of rates of hydrolysis and transglycosylation of chitooligosaccharides. Particularly at low pH the enzyme seems less able to act as transglycosylase. This observation is consistent with the present assumption that AMCase predominantly fulfils a role as food-processing hydrolase in the stomach. The results of our investigation raise intriguing questions about the evolutionary origin and physiological significance of the transglycosidase capacity of chitotriosidase. The human chitotriosidase gene has clearly evolved from a chitinase gene in primitive organisms, and many elements have been conserved among species during evolution (10Boot 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 (345) Google Scholar). It is conceivable, although highly speculative, that transglycosidase activity of the primitive chitinase of the ancestral unicellular organisms allowed biosynthetic formation of increasingly larger chitooligosaccharide polymers near their surface. Prior to the evolution of the more complex, biosynthetic oligosaccharide transferases using activated sugars, the enzyme could thus have played a role in the formation of the first generations of protective coats. An interesting aspect of the transglycosidase activity of chitotriosidase is the ongoing co-formation of chitotetraose while chitin is degraded. Chitotetraose is a structure that recently receives considerable attention. Studies in plants have revealed that chitotetraose may act as signaling molecule that plays important regulatory roles (see for example Ref. 22Bakkers J. Kijne J.W. Spaink H.P. EXS (Basel). 1999; 87: 71-83PubMed Google Scholar for a recent review). Moreover there are reports that chitotetraose may influence embryogenesis in zebra fish (23Bakkers J. Semino C.E. Stroband H. Kijne J.W. Robbins P.W. Spaink H.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7982-7986Crossref PubMed Scopus (69) Google Scholar, 24Semino C.E. Allende M.L. Int. J. Dev. Biol. 2000; 44: 183-193PubMed Google Scholar). In view of this it might be speculated that also in man a challenge with some chitin-containing organism could result in local generation of chitotetraose and subsequently elicit further responses of local cells and the immune system. In this connection it will be of interest to examine more closely in animals the effects of administration of chitotetraose. At present it is generally believed that man completely lacks endogenous chitin and also endogenous substrates for chitinases (see for example Ref. 25Shahabuddin M. Kaslow D.C. Parasitol. Today. 1993; 9: 252-255Abstract Full Text PDF PubMed Scopus (29) Google Scholar). Our study, however, raises the question as to whether endogenous compounds act as (temporary) acceptors for chitobiose moieties transferred by chitotriosidase from chitin-containing infectious organisms. In this manner endogenous materials and cells could become decorated with chitobiose motives. Such motives could act as a marker and play a role in local remodeling events that often occur in sites of infections. In connection with this it will be of great importance to investigate in more detail the potential existence of endogenous acceptors of chitobiose moieties. Such studies should help to reveal whether the transglycosidase activity of chitotriosidase is just an evolutionary remnant or still has a physiological function in man. We thank Daan van Aalten, Sonja van Weely, and Carla Hollak for stimulating discussions and Gabor Linthorst, Marri Verhoek, and Anneke Strijland for technical assistance.