(Received for publication, June 29, 1995; and in revised form, August 25, 1995)
From the
We have recently observed that chitotriosidase, a chitinolytic enzyme, is secreted by activated human macrophages and is markedly elevated in plasma of Gaucher disease patients (Hollak, C. E. M., van Weely, S., van Oers, M. H. J., and Aerts, J. M. F. G.(1994) J. Clin. Invest. 93, 1288-1292). Here, we report on the cloning of the corresponding cDNA. The nucleotide sequence of the cloned cDNA predicts a protein with amino acid sequences identical to those established for purified chitotriosidase. Secretion of active chitotriosidase was obtained after transient transfection of COS-1 cells with the cloned cDNA, confirming its identity as chitotriosidase cDNA. Chitotriosidase contains several regions with high homology to those present in chitinases from different species belonging to family 18 of glycosyl hydrolases. Northern blot analysis shows that expression of chitotriosidase mRNA occurs only at a late stage of differentiation of monocytes to activated macrophages in culture. Our results show that, in contrast to previous beliefs, human macrophages can synthesize a functional chitinase, a highly conserved enzyme with a strongly regulated expression. This enzyme may play a role in the degradation of chitin-containing pathogens and can be used as a marker for specific disease states.
Chitinases are known to occur in a variety of species, including bacteria, fungi, nematodes, plants, insects, and fish(1) . It has often been suggested that man and other mammals do not possess an analogous chitinase(2, 3) . We have recently reported on the existence of a chitinolytic enzyme in man, designated as chitotriosidase, which is secreted in large quantities by activated macrophages(4) . Both N-terminal and internal amino acid sequences of purified chitotriosidase suggest a homology with proteins of the chitinase family, including enzymatically inactive members(5) .
The enzyme chitotriosidase is also of interest for clinical reasons. Chitotriosidase activity is several hundred-fold elevated in plasma of patients suffering from Gaucher disease, a disorder characterized by the presence of large amounts of activated, lipid-laden macrophages in spleen, liver, and other tissues(4) . More modest elevations are found in plasma of patients with sarcoidosis and leishmaniasis(4) . The enzyme can therefore serve as a valuable diagnostic marker particularly for Gaucher disease and may be useful in monitoring the efficacy of therapy. Furthermore, evidence has been obtained that a genetic deficiency of chitotriosidase occurs with high frequency in man; the nature and consequence of this defect is not yet known(4) .
To obtain more insight into the structural features and regulation of the expression of human chitotriosidase, we have cloned the corresponding full-length cDNA. This enabled us to compare the derived primary structure of chitotriosidase with that of other proteins of the chitinase family and to monitor the expression of chitotriosidase mRNA in a model system consisting of cultured human peripheral blood monocytes differentiating into activated macrophages. The results are described in this paper.
A cDNA library was prepared using total RNA from cultured macrophages. About 0.1% of the colonies were positive upon hybridization with the probe. One of them had an insert of 1.7 kilobase pairs, which is large enough to contain the complete coding information for chitotriosidase. The nucleotide sequence of this cloned cDNA revealed an open reading frame of 1398 base pairs long, starting with an ATG codon at position 13 and ending with a TGA codon at position 1410 (Fig. 1). The open reading frame encodes a protein with a characteristic N-terminal 21 amino acids endoplasmic reticulum signal peptide immediately followed by the N-terminal sequence established for the chitotriosidase protein(5) . The cDNA sequence does not indicate the presence of potential N-linked glycosylation sites, which is consistent with the absence of N-linked glycans in isolated chitotriosidase(5) . The predicted protein, after removal of the signal sequence, has a length of 445 amino acids and a calculated molecular mass of 49 kDa, thus corresponding to the larger of the two major isoforms of chitotriosidase isolated previously from Gaucher spleen.
Figure 1: Chitotriosidase cDNA sequence and deduced amino acid sequence. The hydrophobic leader peptide (amino acids 1-21) is underlined. Amino acids in chitotriosidase that are identical to those in at least six out of nine members of the chitinase family are depicted in bold characters. The nine members of the chitinase family used are listed in the legend of Fig. 2, with the exception of Autographa californica chitinase and Nicotiana tabacum chitinase. The stretches of amino acids that are also identified in splenic enzyme are underlined with dots(5) . The putative polyadenylation signal is indicated in bold. The chitotriosidase cDNA sequence has been deposited in the GenBank data base.
Figure 2: Alignment of putative active site regions in members of the chitinase protein family. The proteins are as follows: chitotriosidase and a chitinase of the virus A. californica (GenBank L22858), a chitinase of the tobacco hornworm M. sexta (GenBank U02270), an endochitinase of the nematode B. malayi (GenBank M73689), a human oviduct-specific glycoprotein (GenBank U09550), human cartilage gp-39 (a human glycoprotein produced by chondrocytes and synovial cells (GenBank M80927)), YM-1 (a secretory protein of activated mouse macrophages (Pir S27879)), a chitinase of the fungus Aphanocladium album (SwissProt P32470), a chitinase of the filamentous fungus Trichoderma harzianum (GenBank L14614), chitinase A1 from the prokaryote Bacillus circulans (SwissProt P20533), and a class V chitinase from the plant N. tabacum (GenBank X77110). Residues identical to chitotriosidase are indicated by the reverse type. The human oviductal glycoprotein, human cartilage gp-39, and YM-1 are supposedly not chitinolytic.
A comparison with amino acid sequences of members of the chitinase family, also including the enzymatically inactive proteins, reveals additional homologous regions. In Fig. 1, the amino acids in chitotriosidase that are identical to those in at least six out of nine members of the chitinase protein family are indicated by bold characters.
The predicted C-terminal part of human chitotriosidase shows only homology with two members of this family, Manduca sexta(13) and Brugia malayi chitinases(14) . The homologous C-terminal stretches in these chitinases are characterized by the abundant presence of serine residues that might be modified by glycosylation. In the case of the B. malayi chitinase, O-linked glycosylation has indeed been demonstrated(14) .
Figure 3: Time-related changes in expression of mRNA for chitotriosidase and tartrate-resistant acid phosphatase in monocyte-derived macrophages as monitored by Northern analysis. Human peripheral blood monocytes were isolated and cultured as described under ``Materials and Methods.'' Total RNA was extracted at the time points indicated and analyzed as described under ``Materials and Methods.'' The positions of the 28 S and the 18 S rRNA bands are indicated with arrows. The same blot was used for hybridization with the different probes. The radioactive signals for acid phosphatase (upper graph) and chitotriosidase mRNA (lower graph) were related to that of glyceraldehyde-3-phosphate dehydrogenase mRNA in the same lane (right panels). The level of glyceraldehyde-3-phosphate dehydrogenase mRNA over total RNA was constant in time. The relative intensities of the signals in panels A and B cannot be compared because of differences in the specific activities of the probes.
Several lines of evidence show that the isolated cDNA clone encodes chitotriosidase. First, N-terminal and internal peptide sequences of purified tissue chitotriosidase are present in the protein sequence derived from the cloned cDNA. Second, transfection of COS-1 cells with the cDNA results in the synthesis and secretion of an enzymatically active chitinase that, like splenic chitotriosidase, is inactivated by antiserum raised against the tissue enzyme. Furthermore, we observed that the relative rate of hydrolysis of the artificial substrates 4-methylumbelliferyl-chitotrioside and 4-methylumbelliferyl-chitobioside was identical for tissue chitotriosidase and enzyme produced by COS cells (data not shown). Finally, the absence of N-linked glycans in chitotriosidase is predicted by the cDNA sequence, which is consistent with the results of our previous investigations on purified chitotriosidase (5) .
The results of our study imply that there is a gene in man encoding a chitinolytic enzyme homologous to chitinases from various other species, in particular with respect to the region that is assumed to be an essential part of the catalytic site(15) . Strong homologies between the human enzyme and other chitinases are also noted for various other regions that might be crucial to generate the characteristic conformation of this type of enzyme.
The extensive homology between human and other chitinases is interesting and may have important clinical implications. For instance, the use of parasite chitinase fragments as vaccine targets has been proposed(1, 16) . It may be advisable to use only non-homologous regions of parasite chitinases for vaccination to prevent a potential immunological response to the endogenous human counterpart.
On the basis of its sequence homology with other
chitinases, the human chitotriosidase should be considered to belong to
family 18 of glycosyl hydrolases(17) . It has recently been
proposed that the catalytic core structure of family 18 of glycosyl
hydrolases is an 8-stranded /
(TIM) barrel(18) .
Although some plant endochitinases and lysozyme belong to a family of
glycosyl hydrolases distinct from family 18, we have observed some
similarities between human chitotriosidase and lysozyme(19) .
For instance, chitotriosidase, like lysozyme, is relatively heat stable
and resistant to proteolytic degradation. Chitotriosidase, like
lysozyme, is inhibited in activity by high concentrations of the
substrate 4-methylumbelliferyl-chitotrioside and shows a very broad pH
optimum. Chitotriosidase and lysozyme are both present in lysosomes and
granules of neutrophilic granolocytes. Macrophages constitutively
secrete both chitotriosidase and lysozyme and partly accumulate the
enzymes in their lysosomes. (
)The analogies between
chitotriosidase and other members of family 18 of glycosyl hydrolases,
and some of its similarities with the well characterized lysozyme,
should be helpful in future investigations on structure-function
relationships in chitotriosidase.
Chitotriosidase, unlike most lysosomal hydrolases, is not a house-keeping enzyme. The macrophages are capable of producing very large amounts of the enzyme under specific circumstances. One such condition is the prolonged culture of macrophages derived from peripheral blood monocytes. On the basis of our findings, it may be assumed that chitotriosidase mRNA comprises approximately 0.1% of the total mRNA in macrophages cultured for 3 weeks. Metabolic labeling studies suggest that about 1% of the secretory proteins of these cells is chitotriosidase. Another condition is found in Gaucher disease, in which an inherited deficiency in glucocerebrosidase activity causes lysosomal storage of glucosylceramide in macrophages. This is accompanied by greatly enhanced levels of chitotriosidase in plasma(4) .
The physiological role of the human chitinase still has to be established. On the basis of its known properties, it seems likely to fulfill a role in the degradation of chitin-containing pathogens. The existence of a rather frequent, inherited deficiency in chitotriosidase activity in man and the occurrence of abnormal high enzyme levels in plasma of Gaucher disease patients should help to elucidate the physiological role of chitotriosidase(4) . In this connection, it is interesting to note that plants are known to produce high amounts of chitinases under conditions of stress(20, 21) . The anti-fungal action of these proteins in plants is very well documented(22, 23, 24) . More recently, an additional role in morphogenetic processes in plants has been postulated(12, 21) . The exact function of chitinases in morphogenesis is not clear, since plants are believed to contain no endogenous chitin or analogous structures(21) . It is conceivable that chitotriosidase in man may also have a dual function. Alternatively, homologous proteins may be involved in morphogenetic events in man. The homologous human cartilage gp-39 is a 39-kDa glycoprotein that is produced by articular chondrocytes and synovial cells under specific conditions, as in rheumatoid arthritis. A role for this protein, which lacks chitinolytic activity, has been proposed in tissue remodeling(11) .
The demonstration of the existence of a human chitinase warrants further investigation. The role of chitotriosidase in defense against specific pathogens has to be carefully examined as well as the possibility of another physiological function. Furthermore, the finding that chitotriosidase production is associated with the presence of specifically stressed macrophages may be exploited to monitor progression and correction of a number of pathological conditions in man, such as Gaucher disease, in which these types of cells are involved.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U29615[GenBank].