From the Department of Biochemistry, University of
Amsterdam, Academic Medical Center, 1105 AZ Amsterdam, The
Netherlands and the ¶ Center of Marine Biotechnology, University
of Maryland Biotechnology Institute, Baltimore, Maryland 21202
Received for publication, October 30, 2000
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ABSTRACT |
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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.
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 (1), the microfilarial
sheath of parasitic nematodes (2, 3), and the exoskeleton of all
types of arthropods (4), and in the lining of guts of many insects (5).
Chitinases (EC 3.2.1.14) are
endo- Chitotriosidase is remarkably homologous to chitinases from plants,
bacteria, fungi, nematodes and insects (8, 9). Analogous to some plant
chitinases, recombinant chitotriosidase has been found to inhibit
hyphal growth of chitin-containing fungi such as Candida and
Aspergillus
species.1 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 (7, 13). About 1 in 20 individuals is completely
deficient in enzymatically active chitotriosidase, because of a 24-base
pair duplication in the chitotriosidase gene (14). 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 (14). In
Caucasian populations, up to 35% of all individuals carry this
abnormal chitotriosidase allele and about 5% are homozygous for this
allele (14). 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)2
are reported, and the possible implications of its existence are discussed.
Enzyme Assays--
Chitinase enzyme activity was determined with
the fluorogenic substrates 4-methylumbelliferyl
Crab shell chitin (Poly-
(1-4)- Degradation of Fungal Cell Wall Chitin--
Measurements of
chitin formation during regeneration of fungal spheroplasts was
performed as described by Hector and Braun (16). 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 mM
sodium phosphate, pH 7.5, 1.2 M sorbitol, and 27 mM Purification of the Mouse AMCase--
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 2 M. 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. (17)
using bovine serum albumin as a standard. Fractions containing
chitinase activity were subjected to SDS-PAGE and Western blotting as
described (8). N-terminal protein sequencing was performed as described using a Procise 494 sequencer (Applied Biosystems Perkin Elmer) (8).
Colloidal chitin was prepared as described by Shimahara and Takiguchi
(18).
SDS-PAGE and Glycol-Chitin Gel Electrophoresis--
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 (19), 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 (20).
Isoelectric Focusing--
The native isoelectric point of
chitinases was determined by flat bed isoelectric focusing in
granulated Ultrodex gels (Amersham Pharmacia Biotech) as described
(8).
Northern Blot and RNA Master Blot Analysis--
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 (9). 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 (9). Quantification
of radioactivity was performed using a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA).
cDNA Cloning of the Mouse AMCase--
Reverse transcription
polymerase chain reaction (PCR) fragments were generated from mouse
lung total RNA using degenerate oligonucleotides, as described (9).
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.
cDNA Cloning of the Human AMCase--
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 in COS-1 Cells--
Transient expression of
the various cDNAs in COS-1 cells was performed exactly as described
previously (9).
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-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-1,4-N-acetylglucosaminidases that can fragment
chitin and have been identified in several organisms (6). 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 (7-9). 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 (7). Chitotriosidase is
largely secreted as a 50-kDa active enzyme containing a C-terminal
chitin binding domain (10, 11). 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 (10).
The 50-kDa chitotriosidase form is also synthesized by progenitors of
neutrophilic granulocytes (9) and stored in their specific granules (9,
12).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-N,N'-diacetylchitobiose (4MU-chitobiose; Sigma) and 4-methylumbelliferyl
-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 mM
citric acid, 200 mM sodium phosphate) at the indicated pH.
The standard enzyme activity assay for human chitotriosidase with
4-methylumbelliferyl
-D-N,N',N"-triacetylchitotriose
substrate was performed at pH 5.2, as previously described (7). The
standard AMCase enzyme activity assays with 4MU-chitobiose substrate
were performed at pH 4.5.
-D-N-acetylglucosamine, Sigma) was
used as a natural substrate to determine chitinase activity as
described (10). The chitin fragments were analyzed by
fluorophore-assisted carbohydrate electrophoresis as described by
Jackson (15).
-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).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
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Fig. 1.
Isoelectric focusing profile of chitinolytic
activity in mouse lung extract. Isoelectric focusing was performed
as described under "Experimental Procedures." Chitinolytic activity
was measured using 4MU-chitotrioside substrate. The enzyme activity
present in the different isoelectric focusing fractions is expressed as
a percentage of the total activity present in all fractions.
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.
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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 lack N-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 (21-23). 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.
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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. 4A). 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. 4B).
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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. 5A) and is completely
inactivated by pre-incubation at low pH (Fig. 5B). 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. 5C).
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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.
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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. 8A). 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. (24). 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. 8B).
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The demonstration by Saito et al. (24) 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.
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DISCUSSION |
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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. 6). More recently, it has become clear that mammals also contain such enzymes. Chitotriosidase was the first mammalian chitinase that had been cloned and characterized (7-9). Besides this human phagocyte-specific chitinase, several inactive members of the mammalian chitinase protein family have also been identified. These include oviduct-specific glycoprotein from several mammalian species (reviewed in Refs. 25-27), human HC gp39/YKL-40 (28, 29), mouse BRP39 (30), pig gp38K (31), human YKL-39 (32), and mouse YM1/ECFL/MCRP (33, 34). The functions of these proteins, of which some have been shown to express lectin-like properties (35), are at present unknown. It has been speculated that they might have a role in tissue remodelling processes (28) or chemotaxis (33, 36).
To our knowledge chitotriosidase is the only mammalian chitinase that has been cloned and characterized in detail so far. Our present study describes the discovery of a second acidic mammalian chitinase named AMCase. This enzyme is also able to degrade artificial chitin-like substrates as well as chitin from crab shell and chitin as present in the fungal cell wall.
Sequence homology, conservation of intron-exon boundaries and
chromosomal location suggest that the genes of members of the mammalian
chitinase protein family evolved from a common ancestor by duplication.
This is also suggested by their structural similarities, in particular
between AMCase and human chitotriosidase. Both are members of family 18 of glycosyl hydrolases, showing an 8-stranded /
(TIM)
barrel catalytic core structure (37, 38). Like chitotriosidase, AMCase
contains a N-terminal catalytic core domain of 39 kDa and a C-terminal
chitin binding domain separated by a hinge region (11). An ongoing
crystallographic study on the three-dimensional structures of human
chitotriosidase and AMCase (collaboration with F. Fusetti and B. Dijkstra from the University of Groningen, The Netherlands) should
answer some intriguing questions. For example, the molecular basis for
the profound differences in stability and catalytic capacity at low pH
between the enzymes has to be resolved. It will also be of interest to
establish whether the difference in migration of the two enzymes upon
SDS-PAGE at nonreducing conditions is caused by differences in
disulfide bonds. All 10 cysteines residues in chitotriosidase are
conserved in mouse AMCase. The primary amino acid sequence of mouse
AMCase shows the presence of 2 additional cysteines in the catalytic
core, which are conserved in the human AMCase. Tjoelker et
al. (11) have recently shown that all 6 cysteines in the
chitin-binding domain of human chitotriosidase are involved in
disulfide bonds within this domain and are essential for lectin activity.
In view of our observation that mouse and human AMCase mRNA is highly expressed in the stomach, the noted acidic pH optimum and profound acid stability of AMCase is not surprising. The extreme environment in these parts of the gastrointestinal tract requires such special features. The fact that no AMCase mRNA was detected in the intestine suggests that the protein present in these lower parts of the gastrointestinal tract may originate from the stomach and submaxillary glands. However, AMCase EST clones have been identified in the mouse caecum, tongue, and pancreas recently, indicating that several additional parts of the gastrointestinal tract are involved in the generation of AMCase. Whether the observed chitinase activity in the saliva of patients with periodontal inflammation described by van Steijn et al. (39) can be ascribed to AMCase remains to be established.
We also observed that AMCase mRNA is expressed in the lung (although to a lesser extent than in the stomach) and that enzyme activity is detectable there. At present the exact cellular sources of AMCase are unknown. Recently Guoping et al. (40) identified a silica-induced bronchoalveolar lavage protein with fibroblast growth promoting activity in the rat. This protein is identical to the AMCase we isolated from the rat.3 It has been shown that the protein could be identified in alveolar macrophages of silicotic rats (40), suggesting that at least in the rat lung this enzyme could be generated by macrophages. However, we have been unable to demonstrate any chitinolytic activity in rat alveolar macrophages (not shown). This could indicate that alveolar macrophages are only capable of producing AMCase under a specific stimulus. Moreover, we have also not observed any expression of AMCase in human monocyte-derived macrophages, even under conditions when the cells massively produce chitotriosidase. A detailed characterization of the promoter regions of AMCase and chitotriosidase is required to understand the selective expression of these enzymes. In situ hybridization analysis has to reveal which cells in the respiratory and gastrointestinal tract can express AMCase.
For several vertebrates and invertebrates the presence of chitinase activity in the gastrointestinal tract has been reported (for an overview see Refs. 6, 41, and 42). This activity has sometimes been ascribed to the microorganisms present in the tract. However, gut chitinases have been cloned from several insect species and are thought to be involved in maintenance of the peritrophic matrix (43-45). The peritrophic matrix is a chitinous extracellular layer that surrounds a food bolus in the guts of most arthropods (46), providing a physical barrier to pathogens, facilitating digestion, and protecting against damage by food particles. Our study shows that, at least in rodents and man, a part of the chitinolytic activity found in the gut should be ascribed to an endogenous source also.
The presence of chitinase activity in vertebrates has actually been described earlier, but little is known about the corresponding proteins (6, 42, 47). Place (48) described the purification of a rainbow trout chitinase, which was isolated from the cardiac portion of the stomach. Comparison of the first 26 amino acids of this fish chitinase showed that it is 54% identical to mouse AMCase. Comparison of the complete sequence should reveal more information regarding the evolutionary relationship between the mammalian and fish stomach chitinases.
At present the physiological function of AMCase is unknown. Our study has revealed a remarkable parallel between chitinases and another group of endo-glucosaminidases, the lysozymes. It is well known that distinct lysozyme isoforms occur in various organisms. Lysozymes produced by phagocytes are basic proteins that fulfill a defense function by virtue of their ability to degrade the cell wall of Gram-negative bacteria. In the gastrointestinal tract of some species, acidic lysozymes are expressed that are acid stable and active at low pH. These enzymes are thought to function as food processors (49). By their action, the cell walls of bacteria that ferment plant materials are degraded, allowing the subsequent release and assimilation of their contents. It is conceivable that AMCase also plays a role in food assimilation as earlier proposed for fish chitinases by Lindsay (50), whereas the phagocyte-specific chitotriosidase is primarily involved in defense. The observation that AMCase is also expressed in the lung may point to a dual function for the enzyme, both in defense and food processing.
In ruminant artiodactyls, leaf-eating monkeys, and the bird hoatzin, lysozyme has been adapted by rapid convergent evolution to allow survival and functioning in the acidic, proteolytic environment of the stomach (51). These adaptations changed the global properties of the enzyme by a reduction of the isoelectric point so that the protein is neutral or acidic rather than basic and by a reduction in the number of acid labile bonds and side chains (51). Similar differences can be observed between chitotriosidase and AMCase, suggesting that the same kind of evolutionary processes played a role in chitinase adaptation.
Because AMCase is a functional chitinase, it is conceivable that the existence of AMCase in man has allowed the high panethnic incidence of deficiency in chitotriosidase. It will be of great interest to study also in detail the precise composition of chitinases and their respective functions in lower vertebrates such as fish.
Our demonstration of a novel chitinolytic member of the mammalian
chitinase family that might play an important role in defense and/or
nutrition warrants further investigation. Research on structural properties, regulation of expression, and the evolutionary relationship of the different members of the mammalian chitinase family could give
insights into the physiological role of these interesting proteins.
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ACKNOWLEDGEMENTS |
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We acknowledge Dr. Anton Muijsers, Marri Verhoek, Wilma Donker-Koopman, and Anneke Strijland for skillful technical assistance. Dr. S. van Weely, Dr. D. Speijer, and Dr. D. Blom are acknowledged for helpful comments and suggestions during the preparation of the manuscript. Dr. F. M. Klis, Dr. J. C. Kapteyn, and Dr. G. W. Gooday are acknowledged for helpful suggestions during the studies regarding the fungal cell wall.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Biochemistry, University of Amsterdam, Academic Medical Center, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. Tel.: 31-20-5665161; Fax: 31-20-6915519; E-mail: r.g.boot@amc.uva.nl.
Supported by National Science Foundation Grant IBN-9604265 and
by funds from the Center of Marine Biotechnology, University of
Maryland Biotechnology Institute.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M009886200
1 R. G. Boot, E. F. C. Blommaart, G. W. Gooday, B. A. Friedman, and J. M. F. G. Aerts, manuscript in preparation.
3 R. G. Boot, E. F. C. Blommaart, E. Swart, K. Ghauharali-van der Vlugt, N. Bijl, C. Moe, A. Place, and J. M. F. G. Aerts, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are:
AMCase, acidic
mammalian chitinase;
4MU-chitobiose, 4-methylumbelliferyl
-D-N,N'-diacetylchitobiose;
MES, 4-morpholineethanesulfonic acid;
PAGE, polyacrylamide gel
electrophoresis;
EST, expressed sequence tag;
PCR, polymerase chain
reaction.
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REFERENCES |
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