Marked Differences in Tissue-specific Expression of Chitinases in Mouse and Man
Department of Biochemistry (RGB,APB,MV,JMFGA), and Department of Anatomy & Embryology (PAJdB,AFMM), University of Amsterdam Academic Medical Center, Amsterdam, The Netherlands
Correspondence to: Rolf G. Boot, Department of Biochemistry, University of Amsterdam Academic Medical Center, PO Box 22700, 1100 DE, Amsterdam, The Netherlands. E-mail: r.g.boot{at}amc.uva.nl
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Summary |
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(J Histochem Cytochem 53:12831292, 2005)
Key Words: chitinases acidic mammalian chitinase chitotriosidase gastrointestinal tract macrophage in situ hybridization
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Introduction |
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The physiological function of chitotriosidase is not completely resolved. Its phagocyte-specific expression suggests a role in innate immunity. Highly homologous plant chitinases are prominent "pathogenesis-related proteins" that are induced following attack by pathogens and take part in the defense against chitin-containing fungi (Schlumbaum et al. 1986; Sahai and Manocha 1993
). A similar role for chitotriosidase in the human innate immune system is indicated by our observation that recombinant chitotriosidase is fungistatic in mice models of systemic fungal infections (Stevens et al. 2000
; van Eijk MC, Boot RG, and Aerts JMFG, unpublished data). Interestingly,
1 in 20 individuals is completely deficient in chitotriosidase activity due to a 24-bp duplication in the gene that occurs panethnically (Hollak et al. 1994
; Boot et al. 1998
). This high prevalence of deficiency suggests that human chitotriosidase either no longer fulfills an important function under normal conditions or that other mechanisms may compensate for the lack of a functional chitinase (Boot et al. 1998
). The occurrence of deficiency in chitotriosidase is associated with susceptibility to infection with Wuchereria bancrofti, a filarial parasite whose microfilarial sheath contains chitin (Choi et al. 2001
). In search of compensatory chitinases in mammals we identified and characterized a second mammalian chitinase named acidic mammalian chitinase (AMCase) (Boot et al. 2001
). This 50-kDa enzyme is structurally highly related to chitotriosidase.
AMCase occurs in the gastrointestinal tract and lungs of rodents and man. Suzuki et al. (2002) also detected AMCase mRNA in exocrine cells of the serous type of mice. Unlike human chitotriosidase, AMCase has an acidic pH optimum and is very acid stable (Boot et al. 2001
). The enzyme appears to be adapted to function in the extreme stomach environment, where it may fulfill a role in defense and/or digestion of chitin-containing organisms.
In the lung of mice, but not of man, AMCase is the sole detectable endogenous chitinase. In a number of recent reports the mRNA expression of AMCase in the lung of mice was shown to be highly regulated (Sandler et al. 2003; Xu et al. 2003
; Zhu et al. 2004
; Zimmermann et al. 2004
). Intravenous injection of Schistosoma mansoni eggs was found to cause massive expression of AMCase in the lung of wild-type mice and animals with an exaggerated Th2 response that is dominated by the cytokines IL4 and IL13. This induction did not occur in mice with an exaggerated Th1 response or IL13-knockout mice (Sandler et al. 2003
). Zimmermann et al. (2004)
reported highly induced AMCase mRNA levels in mouse models of experimental asthma induced either by ovalbumin or by Aspergillus fumigatus antigen. This induction was mediated by the STAT6 signaling pathway, again suggesting a role for IL4 or IL13 (Zimmermann et al. 2004
). Very recently it was shown for an aeroallergen asthma mouse model that AMCase is induced in the lung via a Th2-specific, IL13-mediated pathway (Zhu et al. 2004
). Interestingly, AMCase activity appeared instrumental for the pathogenesis of asthma. Inhibition of AMCase, either by a specific antibody or the specific chitinase inhibitor allosamidin, alleviated the Th2-mediated inflammatory damage that occurs in asthma (Zhu et al. 2004
). It has been suggested that inhibition of chitinase activity may render an attractive new therapy for asthma (Couzin 2004
; Zhu et al. 2004
).
So far, all attention in studies with mouse models has been focused on AMCase, whereas chitotriosidase, the dominant chitinase in man, has received little attention. We investigated the expression of chitotriosidase and AMCase in mice in more detail. We show here that remarkable differences between man and mouse exist regarding cell type and tissue-specific expression of chitinases. Comparison of promoter sequences of the human and murine chitinase genes helps to explain the species-specific tissue expression of chitinases. The implications for extrapolating observations on chitinase made in mouse models to the human situation are discussed.
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Materials and Methods |
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RNA Isolation, Northern Blot, and RNA Master Blot Analysis
Total spleen RNA was isolated using the RNAzol B (Biosolve; Barneveld, The Netherlands) RNA isolation kit according to the manufacturer's instructions. For Northern blot analysis, 15-µg samples of total RNA were run in 10 mM Hepes (pH 7.5), 6% formaldehydeagarose gels, transferred to Hybond N nylon membranes (Amersham; Buckinghamshire, UK) by the capillary method, and immobilized by UV cross-linking. Full-length mouse chitotriosidase cDNA was used as probe. Human RNA Master Blots (Clontech; Palo Alto, CA) were used to examine the tissue distribution of the human chitotriosidase transcript according to the instructions of the manufacturer, using the full-length human chitotriosidase cDNA as probe. The probes were radiolabeled with 32P using the random priming method. Hybridization conditions were exactly as previously described (Boot et al. 1995).
Isoelectric Focusing
The native isoelectric point of chitinases was determined by flatbed isoelectric focusing in granulated Ultrodex gels (Pharmacia; Uppsala, Sweden) as described (Renkema et al. 1995).
cDNA Cloning of the Mouse Chitotriosidase
Reverse transcriptionpolymerase chain reaction (RT-PCR) fragments were generated from mouse pooled tissue total RNA using degenerate oligonucleotides, as described (Boot et al. 1995). Obtained fragments were cloned in pGEM-T (Promega; Madison, WI) and sequenced. A comparison with the GenBank mouse expressed sequence tag (EST) database using the basic local alignment search tool (BLAST) at the National Center for Biotechnology Information showed that several EST clones matched the mouse chitotriosidase cDNA sequence. Full-length mouse chitotriosidase cDNA was generated using specific primers based on these deposited sequences. The nucleotide sequence of two independent clones from the PCR were sequenced from both strands by the procedure of Sanger using fluorescent nucleotides on an Applied Biosystems (ABI; Foster City, CA) 377A automated DNA sequencer following ABI protocols.
Transient Expression in COS-1 Cells
Transient expression of the various cDNAs in COS-1 cells was performed to generate recombinant enzyme exactly as described previously (Boot et al. 1995).
Tissue Processing
More detailed practical protocols for fixation, paraffin embedding, mounting, and sectioning have been described (Moorman et al. 2000). In short, tissues removed from a FVB mouse were fixed for 4 hr to overnight in freshly prepared 4% formaldehyde in PBS by rocking at 4C. The tissues were then dehydrated in a graded ethanol series, paraffin embedded, cut into sections, and carefully mounted on aminoalkylsilane-coated slides to prevent loss of the tissue sections during the extensive treatments of the in situ hybridization (ISH) procedure.
RNA Probes and Probe Specification
Digoxigenin-labeled probes were made according to the manufacturer's specifications (Roche; Mannheim, Germany). RNA probes complementary to the full-length mouse mRNAs encoding chitotriosidase, AMCase, GOB-5 or calcium-activated chloride channel 3, glutamine synthase, and lysozyme P were used. Due to the high identity, the lysozyme probe under the conditions used also detects the lysozyme M mRNA.
Non-radioactive ISH
Non-radioactive ISH was performed as described (Moorman et al. 2001). In short, after removal of paraffin, sections were pretreated by proteolytic digestion for 515 min at 37C with 20 µg/ml proteinase K dissolved in PBS, followed by a 5-min rinse in 0.2% glycine/PBS, and two rinses of 5 min in PBS. Sections were then re-fixed for 20 min in 4% formaldehyde/0.2% glutaraldehyde dissolved in PBS to ensure firm attachment of the sections to the microscope slides and washed twice in PBS for 5 min. Sections were prehybridized in hybridization mix without probe for 1 hr at 70C and then hybridized overnight at 70C. The hybridization mixture was composed of 50% formamide, 5x SSC, 1% block solution (Roche), 5 mM EDTA, 0.1% Tween-20, 0.1% Chaps (Sigma), 0.1 mg/ml heparin (Becton-Dickinson; Mountain View, CA), and 1 mg/ml yeast total RNA (Roche). Probe concentration was
1 ng/µl. Approximately 6 µl hybridization mix was applied to the sections, and no coverslips were used. After hybridization, sections were rinsed in 2x SSC, pH 4.5, washed three times for 30 min at 65C in 50% formamide/2x SSC, pH 4.5, followed by three 5-min washes in PBST. Probe bound to the section was immunologically detected using sheep anti-digoxigenin Fab fragment covalently coupled to alkaline phosphatase and NBT/BCIP as chromogenic substrate, essentially according to the manufacturer's protocol (Roche). Sections were washed with double-distilled water, dehydrated in a graded ethanol series and xylene, and embedded in Entellan (Merck; Darmstadt, Germany).
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Results |
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Alignment of mouse and human chitotriosidase amino acid sequences revealed that there is an identity of 75% and a similarity of 78%. To group all the known mouse and human chitinase protein family members and to determine whether this cloned sequence is the true ortholog of human chitotriosidase, multiple sequence alignments of their cDNAs, coding for the catalytic 39-kDa domain only, were made using the Clustal X program (Thompson et al. 1997). The sequences used for this alignment are human chitotriosidase (GenBank Accession Number: U29615), human-HC gp39 (GenBank Accession Number: M80927), human-AMCase (GenBank Accession Number: AF290004), human-oviductin (GenBank Accession Number: U09550), human-YKL39 (GenBank Accession Number: U49835), mouse-BRP39 (GenBank Accession Number: X93035), mouse-YM1 (GenBank Accession Number: M94584), mouse-AMCase (GenBank Accession Number: AF290003), and mouse-oviductin (GenBank Accession Number: D32137). Grouping of these members is calculated by the method of Neighbor Joining by Saitou and Nei (Saitou and Nei 1987
). This analysis showed that the cloned mouse sequence is grouped together with human chitotriosidase. It suggests that the mouse sequence is more homologous to human chitotriosidase than to any other member of the chitinase protein family, which is indicative of being orthologs of each other.
Using the Mouse Genome Server from the Mouse Genome Sequencing Consortium (at http://www.ensembl.org/Mus_musculus), we identified genomic sequences that contained the chitotriosidase gene, enabling the determination of intronexon boundaries. The boundaries are all conserved between human and mouse chitotriosidase genes (data not shown). Moreover, it was found that the mouse chitotriosidase gene is located on chromosome 1 band E4 near the boundary with F. This region is syntenic with human chromosome 1q32. On human 1q32, only two genes of members of the chitinase protein family are located, namely, chitotriosidase (CHIT1) and HC gp39 (CHI3L1) (Jin et al. 1998). On mouse chromosome 1 band E4 the mouse BRP39 (Chi3l1), the mouse ortholog of HC gp39, was already identified (Jin et al. 1998
). The data present on the Mouse Genome Server show that, in addition to the BRP39 gene, the mouse chitotriosidase gene is also found at this locus (see Figure 1 for overview). In addition to the syntenic chromosomal locations, the homology in intronic sequences of the mouse and human chitotriosidase genes indicate that they are orthologs.
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Using ISH, no mRNA for chitotriosidase and AMCase was detectable in brain, colon, pancreas, liver, heart, kidney, skin, and spleen (data not shown) in agreement with the results obtained with the Northern blot analysis. The expression of chitinases in the gastrointestinal tract of mice is complex. Starting in the mouth, profound expression of both chitinases is observed. Chitotriosidase expression was mainly detected in the mucosal surface of the tongue in the stratified squamous epithelium of the papilla (Figures 4A4E). Whereas AMCase is mainly found in the specialized minor lingual salivary glands of the serous type, the so-called glands of von Ebner (Figures 4F and 4G), in agreement with the results described (Suzuki et al. 2002; Goto et al. 2003
), in the major salivary glands intense expression of AMCase was observed in the parotid gland (Figures 5A5C), and no expression of chitotriosidase was noted (data not shown).
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The expression of chitinases in the lung is of particular interest. In normal human lung, chitotriosidase is prominently expressed by alveolar macrophages. In sharp contrast, we were unable to detect significant amounts of chitotriosidase mRNA in murine lung. On the other hand, AMCase mRNA is expressed by alveolar macrophages in the mouse (Figure 6).
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The remarkable difference in expression of mouse and human chitotriosidase was further investigated by analysis of the promoter regions. Mouse EST cDNA sequences containing the 5' untranslated region indicate that the mouse chitotriosidase gene has an extra exon compared with the human gene. This first exon is located at least 7000 bp upstream. The human chitotriosidase promoter is homologous to a region found in mouse intron 1 just upstream of exon 2. In conclusion, the promoter of the mouse chitotriosidase gene differs fundamentally from the human one (Figure 7). This likely explains the noted differences in cell type expression of human and mouse chitotriosidase.
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Discussion |
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The acid-stable AMCase is produced predominantly in the glandular portion of the stomach, at the bottom of the gastric glands in chief cells, close to the acid-producing parietal cells, consistent with its features. Stomach lysozymes from ruminants have adapted to function optimally in the hostile acid environment by subtle changes in amino acid composition. The same is also the case for the mouse AMCase that has also adapted to function in the stomach. Analysis of the three-dimensional structure of chitotriosidase and AMCase should reveal the key amino acid substitutions required for the adaptation. The available information on the structure of chitotriosidase should assist further investigations on this matter (Fusetti et al. 2002).
The human AMCase is less well equipped to function in a highly acidic environment compared with the mouse enzyme. Differences in physiology or diet may have contributed to this. Live insects are an important component in the diet of wild mice (Landry 1970).
Intriguingly, in the cow AMCase is produced and secreted by the liver and is present in large amounts in serum (Suzuki et al. 2001). The function of the enzyme is unclear. It seems likely that expression is driven by yet another type of promoter, adding to the rapidly growing complexity of the mammalian chitinase protein family.
Overexpression of chitinases occurs in a number of pathologies in mouse and man. Chitotriosidase is the dominant chitinase in man that is highly expressed in specific cell types including tissue macrophages. In various disorders in which activated macrophages are implicated, elevated plasma chitotriosidase levels occur, e.g., lysosomal lipid storage disorders, sarcoidosis, visceral Leishmaniasis, extended atherosclerosis such as Tangier disease, and thalassemia (Hollak et al. 1994; Barone et al. 1999
; Boot et al. 1999
; Grosso et al. 2004
). In sharp contrast, in mice, expression of chitotriosidase is confined to the gastrointestinal tract and AMCase seems to be the sole endogenous chitinase in tissues such as lung. These species differences seem to be due to distinct promoters of the chitotriosidase genes in mouse and man. The mouse chitotriosidase transcription start site is located far upstream compared with the human situation because the mouse gene contains an extra exon at the 5' end. Homology is noted between the human promoter and a region in intron 1 of the mouse gene, just upstream of the second exon. Whether this region can indeed act as an alternative promoter for the mouse chitotriosidase gene is at present not known and a topic of further investigation.
The promoter regions of the mouse and human AMCase genes are relatively comparable.
In contrast to Suzuki and coworkers (2002), we were able to detect AMCase mRNA in murine lung with ISH, confirming our Northern blot analysis (Boot et al. 2001
). This discrepancy might be due to differences in mice strains or to the sensitivity of the ISH methods. The expression of AMCase mRNA is found to be induced in the lung of mice under various pathological conditions, most strikingly in Th2-driven asthma models (Sandler et al. 2003
; Xu et al. 2003
; Zhu et al. 2004
; Zimmermann et al. 2004
). Prominently increased expression of AMCase has also been observed in lung biopsies from asthmatic patients (Zhu et al. 2004
).
A surprising role has recently been ascribed to chitinases in the pathogenesis of asthma. It was found that inhibition of chitinase activity in lungs of asthmatic mice alleviates the inflammatory pathology (Zhu et al. 2004). This opens a potential new avenue for therapeutic intervention, i.e., the use of specific chitinase inhibitors such as allosamidin (Zhu et al. 2004
). However, extrapolation of the findings with mice to man is complicated by the fact that it is presently unclear whether in addition to AMCase, chitotriosidase is also overexpressed in lung of asthmatic patients. It has been shown that in sarcoidosis, a systemic granulomatous disease, chitotriosidase is elevated in plasma and bronchoalveolar lavage (Hollak et al. 1994
; Grosso et al. 2004
). It is therefore unclear whether inhibition of both AMCase and chitotriosidase would be required for intervention in the inflammatory process in lungs of asthmatic patients. It may also be possible that selective inhibition of AMCase is more desirable. In this regard, it will be of interest to study whether the relatively common chitotriosidase deficiency influences the clinical course of asthma.
In conclusion, the remarkable differences in cell type and tissue-specific expression of chitotriosidase in man and mouse seem to be mediated by usage of different promoter regions, gene duplications, and different environmental pressures during the evolution of these different species. Further research is required regarding the physiological functions of the chitinase and their potentially harmful role in excessive inflammatory responses.
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Acknowledgments |
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Footnotes |
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