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(Received for publication, July 25, 1994; and in revised form, October 3, 1994) From the
Recently we noted (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) that the clinical manifestation of Gaucher disease is
associated with a several hundred-fold increase in chitotriosidase
activity in plasma. We report on the purification and characterization
of the protein. Two major isoforms of chitotriosidase with
isoelectric points of 7.2 and 8.0 and molecular masses of 50 and 39
kDa, respectively, were purified from the spleen of a Gaucher patient.
The N-terminal amino acid sequence of the two forms proved to be
identical. An antiserum raised against the purified 39-kDa
chitotriosidase precipitated all isozymes. Chitotriosidase activity was
earlier found to be completely absent in some individuals. These
findings in combination suggest that a single gene may encode the
different isoforms of chitotriosidase. Both the N-terminal sequence
and an internal sequence chitotriosidase proved to be homologous to
sequences in proteins that are members of the chitinase family (Hakala,
B. E., White, C., and Recklies, A. D. (1993) J. Biol. Chem. 268, 25803-25810). The human chitotriosidase described here
showed chitinolytic activity toward artificial substrates as well as
chitin and may therefore be considered to be a chitinase. Gaucher disease is a recessively inherited lysosomal storage
disorder in which the activity of the enzyme glucocerebrosidase is
markedly decreased. This results in accumulation of the glycolipid
glucosylceramide in the lysosomes of macrophages(1) . Recently,
enzyme-replacement therapy has been successfully applied by infusing
purified placental glucocerebrosidase, which has been modified so as to
target the enzyme to macrophages(2) . The clinical
manifestation of Gaucher disease is generally accompanied by increased
plasma levels of certain enzyme activities, including acid phosphatase
5B(3) , angiotensin-converting enzyme(4, 5) ,
lysosomal hydrolases(6, 7) , and
lysozyme(5, 8) . For instance, there is an
approximately 10-fold increase in the activity of acid phosphatase 5B
in plasma of Gaucher patients compared with that of controls (see e.g.(9) ); the elevations in the activity of other
enzymes are much less pronounced. Chitotriosidase activity was found
to be on average more than 600 times increased in plasma of Gaucher
patients compared with controls(9) . Such a marked elevation
has, so far, been observed only in samples from Gaucher patients and
not in plasma from patients with other pathological conditions.
Moreover, successful therapeutic intervention in Gaucher disease proved
to be accompanied by a rapid and marked reduction in the
chitotriosidase levels in plasma(9) . In our previous study it
was observed that chitotriosidase is a secretory protein of cultured
macrophages(9) . A small amount of enzyme is also found
intracellularly, possibly in lysosomes. The enzyme does not show the
characteristic acid pH optimum of lysosomal enzymes but has very
similar activity in the pH range 3-8. Human chitotriosidase
had not been purified so far, and little is known about the nature and
function of the enzyme. Furthermore, the relationship between the
several hundred-fold increased plasma levels of chitotriosidase and the
pathophysiology of Gaucher disease is unclear. Here, we report on the
purification of chitotriosidase from Gaucher spleen and describe a
number of characteristics of the enzyme.
The two PNP substrates (Sigma, p-nitrophenyl
Chitinase activity was determined using chitin azure
(Sigma), which was suspended in McIlvain buffer (pH 5.2). The final
concentration of chitin azure particles was 10 mg/ml. Degradation was
monitored by spectrophotometric detection at 550 nm of soluble azure
after centrifugation(10) . Chitinase from Serratia
marcescens (Sigma) was used as a control. Lysozyme activity was
determined according to Mörsky (11) by
measuring the decrease in absorbance at 450 nm of a Micrococcus
lysodeikticus suspension (Sigma, 0.26 mg/ml) in McIlvain buffer
(pH 5.2). Lysozyme from human milk (Sigma) was used as a control.
Protein concentrations were determined according to the method of
Lowry et al.(12) , using bovine serum albumin as
standard.
Figure 1:
Analysis of protein
constituents of fractions of a typical chitotriosidase purification
procedure. Proteins were separated on 12.5% SDS-PAGE gel and visualized
by silver staining. Molecular mass standards are indicated (kDa). Lane1, spleen extract; lane2,
pool of PBE column; lane3, pool of Sephadex G-100
column; lane4, IEF fraction with pH of 8.0; lane5, IEF fraction with pH of 7.2. The 39-kDa isoform of
chitotriosidase is indicated by an arrow and the 50-kDa
isoform by an arrowhead.
The native molecular
masses of the pI 8.0 and 7.2 chitotriosidases were 29 and 37 kDa,
respectively, on a calibrated Sephadex G-100 column (not shown). Table 1gives the results overview of a typical isolation. The
amount of 39-kDa chitotriosidase in the final pI 8.0 fraction was
determined by silver staining and comparison with known amounts of
bovine serum albumin. The isolation procedure resulted in a more than
3600-fold purification of the 39-kDa (pI 8.0) chitotriosidase from an
extract of a spleen from a type I Gaucher patient. Four independent
isolations gave comparable results.
Figure 2:
N-terminal amino acid sequence and an
internal amino acid sequence of chitotriosidase; alignment with members
of the chitinase protein family. The N-terminal sequence was determined
for both the 39- and 50-kDa isoforms of chitotriosidase and proved to
be identical. The internal sequence was obtained from a tryptic
fragment of the 39-kDa isoform of chitotriosidase. The proteins are: HC
gp-39, a human glycoprotein produced by chondrocytes and synovial cells
(GenBank M80927); a bovine oviduct-specific glycoprotein (GenBank
D16639); a protein secreted in bovine whey during involution (SwissProt
P30922; only the N-terminal amino acid sequence of this protein is
available); YM-1, a secretory protein of activated mouse macrophages
(Pir S27879); an endochitinase of the nematode B. malayi (SwissProt P29030); a chitinase of the hornworm Manduca sexta (GenBank U02270); and a chitinase of the fungus A. album (SwissProt P32470). Residues identical to chitotriosidase are
indicated by whiteletters; capitalletters indicate residues with similar properties to
those in chitotriosidase.
Homology was proven to exist between the N-terminal
and internal sequences of the human chitotriosidase and those of
proteins that are members of a recently recognized chitinase protein
family(14) , as shown in Fig. 2. This family consists of
proteins from various organisms, with strong homology in several
domains including the region that is involved in the catalysis of the
hydrolysis of chitin and the artificial substrate
4MU-chitotrioside(15) .
Figure 3:
Isoelectric focusing profiles of
chitotriosidase activity in Gaucher materials. Isoelectric focusing was
performed as described under ``Materials and Methods.''
Chitotriosidase activity was measured with the 4MU-chitotrioside
substrate. A, Gaucher spleen extract; B, Gaucher
plasma sample.
To study the relationship between the various
chitotriosidase isozymes, an antiserum was raised in a rabbit against
purified native 39-kDa enzyme. This antibody recognized only native
chitotriosidase, and more than 98% of the chitotriosidase activity in
the Gaucher spleen extract was immunoprecipitable with this immobilized
anti-(39-kDa chitotriosidase) antiserum. Chitotriosidase in pI 8.0,
7.2, and 5.5-6.0 fractions was identically precipitated in
immunotitration experiments (not shown). Earlier we found that some
individuals are deficient in plasma chitotriosidase
activity(9) . We observed that a deficiency in plasma was
accompanied by a deficiency in other materials, such as spleen. The
deficiency was not due to the presence of some inhibitor but probably
the result of some inherited defect. These observations suggest that
the different chitotriosidase isozymes are most likely encoded by a
single gene. All lysosomal hydrolases, with the exception of
lysozyme, contain N-linked glycans that bind strongly to
either the lectin concanavalin A or the lectin Ricinus communis agglutinin. When tested, chitotriosidase showed no affinity for
binding to these two lectins (not shown). Incubation of pure 39-kDa
chitotriosidase with endoglycosidases H and F or N-glycanase
also did not result in a change in apparent molecular mass.
Furthermore, preliminary results of metabolic labeling experiments with
cultured macrophages revealed no shift in mobility upon addition to the
culture medium of tunicamycin (not shown), again suggesting the absence
of N-linked glycosylation.
Since 4MU-chitotrioside has been
reported to be a substrate for lysozyme(16) , the activity was
studied of purified chitotriosidase toward a suspension of cell walls
of M. lysodeikticus, a natural substrate for lysozyme.
Purified chitotriosidase showed no lysozyme activity, as shown in Table 2. Because of the high degree of homology of
chitotriosidase with a number of chitinases, it was of interest to
study the capacity of chitotriosidase to degrade chitin, a polymer of
In this report we describe the purification and partial
characterization of the newly discovered human chitotriosidase that is
highly elevated in Gaucher patients(9) . The chitotriosidase
characterized by us may be identical to a human plasma
4-methylumbelliferyl-tetra-N-acetylchitotetraose hydrolase
described by Den Tandt and co-workers(17, 18) . These
investigators found that their partially purified enzyme did not
exhibit hyaluronidase, neutral endoglucosaminidase,
aspartylglucosaminidase, We noted that the human chitotriosidase was
still active at 50 °C and could be inhibited by
Ag Our finding that chitotriosidase is a chitinase is of
particular importance since, even in recent publications (see e.g.(22) ), the human body is still believed to contain no
chitin. Recently it has been recognized that not only do chitinases
from various non-mammalian organisms (such as bacteria, fungi, plants,
and insects) share structural homology but proteins with a partially
similar structure also occur in mammals. The members of the so-called
chitinase protein family (14) differ in ability to catalyze the
hydrolysis of chitin or chitin-like substrates such as
4-methylumbelliferyl chitotrioside. All documented mammalian members of
the family have been found, so far, to be without chitinolytic
activity. These mammalian proteins include a human cartilage protein
(HC gp-39)(14, 23) , a murine protein secreted by
activated macrophages (YM-1; only documented in the Pir data bank), a
bovine whey protein (24) , as well as a baboon (25) and
a bovine oviduct-specific glycoprotein(26) . Their inability to
hydrolyze substrate is most likely explained by the absence of critical
acidic amino acids in the catalytic site region(15) , as can be
deduced from the nucleotide sequence of cDNA encoding HC gp-39, YM-1,
and bovine oviduct-specific glycoprotein. The chitotriosidase isolated
from Gaucher spleen clearly differed from the other mammalian members
of the chitinase protein family. This protein appears to be more
closely related to the chitinases of non-mammalian organisms, since it
is also a functional chitinolytic enzyme. The human chitotriosidase
described here may be involved in defense against and in degradation of
chitin-containing pathogens such as fungi, nematodes, and insects. The
function of the members of the chitinase protein family without
chitinase activity is unknown. Some, such as HC gp-39 and the bovine
whey protein, are expressed in association with remodeling
events(14) . Interestingly, in plants, chitinases are believed
to be involved in defense against pathogens as well as morphogenetic
processes that involve remodeling (see (21) , and references
therein). The role of the chitinases in morphogenesis is poorly
understood since plants do not contain endogenous chitin. It cannot be
excluded that, in analogy to the situation in plants, chitotriosidase
in man also fulfills multiple functions. The relationship of the
various chitotriosidase isozymes that occur in man is not precisely
understood. However, the finding that all chitotriosidase activity is
absent in some individuals (9) suggests that this enzyme is
encoded by a single gene. This suggestion is in agreement with our
present finding that an antibody raised against the 39-kDa
chitotriosidase precipitated all isozymes. Moreover, the N terminus of
at least the 39- and 50-kDa isozymes was identical. The heterogeneity
in chitotriosidase could therefore be due to alternative splicing,
post-translational proteolytic processing, or differences in
glycosylation. Further information about the structure, the
regulation of synthesis, and the routing of human chitotriosidase as
well as its physiological substrate is required in order to be able to
understand the role of the enzyme under normal and pathological
conditions. Cloning of the corresponding cDNA and analysis of the
processing of the protein are, therefore, being undertaken. These
investigations will be crucial to the identification of possible
effects of the relatively common deficiency in enzyme activity in man (9) and to identify the cause and consequences of the strong
increase in plasma levels of chitotriosidase in clinically affected
Gaucher patients.
Volume 270,
Number 5,
Issue of February 3, 1995 pp. 2198-2202
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Enzyme Assays
Chitotriosidase and chitobiosidase
activities were routinely determined with the fluorogenic substrates
4MU(
)-chitobiose (Sigma, 4-methylumbelliferyl
-D-N,N`-diacetylchitobiose) and
4MU-chitotriose (Sigma, 4-methylumbelliferyl
-D-N,N`,N"-triacetylchitotriose).
Substrate mixtures contained 0.027 mM substrate and 1 mg/ml
bovine serum albumin in McIlvain buffer (100 mM citric acid,
200 mM sodium phosphate), pH 5.2. Assays were performed as
described previously(9) .-D-N,N`-diacetylchitobiose and p-nitrophenyl
-D-N,N`,N"-triacetylchitotriose)
were used in McIlvain buffer (pH 5.2) at a concentration of 370 and 270
µM, respectively. Assays (final volume, 100 µl) were
stopped with 50 µl of 3 M glycine-NaOH buffer (pH 10.6).
The p-nitrophenyl formed was determined spectrophotometrically
at 405 nm.Purification of Chitotriosidase
Detergent-free
spleen extract was prepared by homogenization of Gaucher type I spleen
in 4 volumes of water, using an Ultra-turrax and centrifugation for 20
min at 15,000 
g. The pH of the supernatant was
adjusted to 8.5 using 1 M Tris buffer (final concentration, 25
mM), and the supernatant was applied to a polybuffer exchange
column (PBE 94, Pharmacia Biotech Inc.); the column was equilibrated
and eluted with 25 mM Tris buffer (pH 8.5). Breakthrough
fractions with highest chitotriosidase activity were pooled and
concentrated by Amicon PM10 ultrafiltration. This pool was applied to a
Sephadex G-100 (Pharmacia) column and eluted with 25 mM Tris
buffer (pH 8.0). Fractions were collected and peak fractions containing
enzyme activity were pooled and concentrated again. As a final step in
the isolation procedure preparative isoelectric focusing was performed. Isoelectric Focusing
Preparative flat-bed
isoelectric focusing was performed using Ultrodex (Pharmacia)
containing 0.5% (v/v) Triton X-100 and 0.1% w/v ampholytes (Servalyte
4-9, Serva). Focusing was performed overnight at 10 °C at 400
V, using an LKB 2117 Multiphor apparatus as described by the
manufacturer. The gel was fractionated and extracted with water, after
which the chitotriosidase activity and pH of the fractions were
determined.Chitotriosidase Fragmentation by Proteolytic
Digestion
Purified chitotriosidase was denatured by boiling in
1% -mercaptoethanol and 0.5% SDS. Digestion was done with trypsin
(Boehringer Mannheim) (chitotriosidase:trypsin, w/w, was about 100:1)
at room temperature for 5 min and stopped by boiling in SDS-PAGE sample
buffer.SDS-PAGE
SDS-PAGE was performed on a Pharmacia
Phast-gel system, according to the manufacturer's instructions,
using 12.5% (w/v) acrylamide gels. After electrophoretic separation the
gels were silver-stained. For the separation of proteins prior to
sequencing, 10% SDS-PAGE gels were used according to the method of
Laemmli(13) . Protein digests were separated on 12% gels.Protein Sequencing
Protein samples were separated
on SDS-PAGE and blotted to polyvinylidene difluoride membrane (Bio-Rad)
using a blotting buffer containing 50 mM Tris, 50 mM borate, 20% (v/v) methanol, and 0.02% (w/v) SDS (pH
8.1-8.5). Blots were stained with 1% (w/v) Coomassie Brilliant
Blue (R-250) in 50% v/v methanol, destained with 10% acetic acid in 50%
methanol, and dried. Protein bands were applied to a Beckman/Porton LF
3000 protein sequencer coupled to a Beckman System Gold
phenylthiohydantoin analyzer. The sequences obtained were compared with
those present in the EMBL data bank.Determination of Native Molecular Weight by Gel
Filtration
Sephadex G-100 (Pharmacia) gel filtration was used to
determine the native molecular weight of chitotriosidase. A column was
calibrated using the following proteins as standards: cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), ovalbumin (43 kDa),
bovine serum albumin (67 kDa), and alcohol dehydrogenase (150 kDa).Immunoprecipitation
Immunoprecipitation of
chitotriosidase with immobilized antibodies was performed as described
in (9) .
Purification of Chitotriosidases
Chitotriosidase
activity was found to be about 50-fold increased in spleen of a type I
Gaucher patient as compared with spleen from a control subject. The
enzyme activity was completely recovered in the supernatant of a
detergent-free extract that was used as starting material for the
isolation of chitotriosidase. The extract was applied on a polybuffer
exchange chromatofocusing column (PBE 94), equilibrated to pH 8.5. The
chitotriosidase activity was not bound and only slightly retained by
the column, in contrast to total protein. The fractions enriched in
enzyme activity were pooled and, after concentration via
ultrafiltration, subjected to Sephadex G-100 gel filtration. Fractions
containing chitotriosidase activity were pooled, concentrated, and
subjected to isoelectric focusing. Measurement of chitotriosidase
activity in the fractionated gel showed the presence of two distinct
forms of chitotriosidase with apparent pIs of 7.2 and 8.0, respectively
(not shown). Analysis by SDS-PAGE and silver staining showed that the
pI 8.0 fraction contained a protein with an apparent molecular mass of
39 kDa (Fig. 1, lane4). Depending on the
fractionation of the gel, minor amounts of other proteins were
sometimes noted in chitotriosidase-containing fractions in the pI range
7.9-8.2. However, chitotriosidase activity in such fractions
always correlated with the concentration of the 39-kDa protein only.
The chitotriosidase-containing fractions with pI around 7.2 contained
several proteins with apparent molecular masses of 50, 42, 25, and 18
kDa (Fig. 1, lane5).
Amino Acid Sequences of Chitotriosidases
The
sequence of the first 22 N-terminal amino acids of the 39-kDa protein
with pI 8.0 is presented in Fig. 2. In the fraction containing
chitotriosidase activity with pI of about 7.2, only the 50-kDa protein
could be sequenced. The first 22 amino acids at the N terminus of this
protein were identical to those at the N terminus of the 39-kDa (pI
8.0) protein. Digestion of the purified 39-kDa chitotriosidase with
trypsin resulted in a characteristic pattern of fragments. An internal
sequence of 21 amino acids was obtained from a digestion fragment of
30-kDa protein.
Chitotriosidase Isozymes
Besides the two
predominant forms of chitotriosidase with pI 7.2 and 8.0, isoelectric
focusing of several Gaucher spleen extracts revealed minor forms of pI
5.5-6.0 and pI about 6.5 (Fig. 3A). The apparent
isoelectric point of chitotriosidase activity in Gaucher plasma was
predominantly 7.2, with minor amounts of 6.0 and 8.0 (Fig. 3B). The isoelectric focusing profiles of
chitotriosidase in corresponding control materials were comparable (not
shown).
Hydrolysis of Substrates
The results of
experiments on the substrate specificity of chitotriosidase are shown
in Table 2. Purified samples of different pI forms showed a
higher activity toward 4MU-chitotrioside substrate than toward
4MU-chitobioside, the ratio of chitobioside/chitotrioside activity of
all chitotriosidase preparations being about 0.7. Both PNP-chitobioside
and PNP-chitotrioside were hydrolyzed by purified chitotriosidase.
However, in the case of the PNP-substrates the chitobioside substrate
was more rapidly hydrolyzed.
-1,4-linked N-acetylglucosamine moieties. Chitin azure
was used as substrate. Table 2shows that chitin azure was,
indeed, a substrate for this enzyme. When related to the hydrolysis of
4MU-chitotrioside, degradation of chitin azure by the human
chitotriosidase was even better than by the bacterial chitinase
studied.
-hexosaminidase, -glucosidase, or
chitobiase activity. We, too, were unable to demonstrate any
-hexosaminidase or -glucosidase activity for the purified
chitotriosidase. Nor was the enzyme able to hydrolyze the
-1-4 linkage between N-acetylglucosamine and
muramic acid in cell walls from M. lysodeikticus, and thus it
clearly differs from lysozyme. The relatively high enzymatic activity
toward chitin suggests that our human chitotriosidase may be considered
to be a functional chitinase. Indeed, sequencing of the N terminus and
a digestion fragment of purified human chitotriosidase revealed that
this protein shares homology with chitinases from non-mammalian
organisms, e.g. the nematode Brugia malayi(19) or the fungus Aphanocladium
album(20) .. Similar properties have been documented for the
chitinase (Ch1) of A. album (see (21) , and references
therein).
)
We thank Sarah Jones, Marijn van der Neut Kolfschoten,
and Jessica Teeling for their skillful assistance. Dr. J. M. Tager and
Dr. S. van Weely are acknowledged for their helpful comments and
suggestions during the preparation of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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F. Fusetti, H. von Moeller, D. Houston, H. J. Rozeboom, B. W. Dijkstra, R. G. Boot, J. M. F. G. Aerts, and D. M. F. van Aalten Structure of Human Chitotriosidase. IMPLICATIONS FOR SPECIFIC INHIBITOR DESIGN AND FUNCTION OF MAMMALIAN CHITINASE-LIKE LECTINS J. Biol. Chem., July 5, 2002; 277(28): 25537 - 25544. [Abstract] [Full Text] [PDF]
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L. W. Tjoelker, L. Gosting, S. Frey, C. L. Hunter, H. Le Trong, B. Steiner, H. Brammer, and P. W. Gray Structural and Functional Definition of the Human Chitinase Chitin-binding Domain J. Biol. Chem., January 7, 2000; 275(1): 514 - 520. [Abstract] [Full Text] [PDF]
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 R. G. Boot, T. A. E. van Achterberg, B. E. van Aken, G. H. Renkema, M. J. H. M. Jacobs, J. M. F. G. Aerts, and C. J. M. de Vries Strong Induction of Members of the Chitinase Family of Proteins in Atherosclerosis : Chitotriosidase and Human Cartilage gp-39 Expressed in Lesion Macrophages Arterioscler. Thromb. Vasc. Biol., March 1, 1999; 19(3): 687 - 694. [Abstract] [Full Text] [PDF]
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R. G. Boot, G. H. Renkema, M. Verhoek, A. Strijland, J. Bliek, T. M. A. M. O. de Meulemeester, M. M. A. M. Mannens, and J. M. F. G. Aerts The Human Chitotriosidase Gene. NATURE OF INHERITED ENZYME DEFICIENCY J. Biol. Chem., October 2, 1998; 273(40): 25680 - 25685. [Abstract] [Full Text] [PDF]
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N. O. Keyhani and S. Roseman The Chitin Catabolic Cascade in the Marine Bacterium Vibrio furnissii. MOLECULAR CLONING, ISOLATION, AND CHARACTERIZATION OF A PERIPLASMIC CHITODEXTRINASE J. Biol. Chem., December 27, 1996; 271(52): 33414 - 33424. [Abstract] [Full Text] [PDF]
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B. Hu, K. Trinh, W. F. Figueira, and P. A. Price Isolation and Sequence of a Novel Human Chondrocyte Protein Related to Mammalian Members of the Chitinase Protein Family J. Biol. Chem., August 9, 1996; 271(32): 19415 - 19420. [Abstract] [Full Text] [PDF]
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R. Adam, B. Kaltmann, W. Rudin, T. Friedrich, T. Marti, and R. Lucius Identification of Chitinase as the Immunodominant Filarial Antigen Recognized by Sera of Vaccinated Rodents J. Biol. Chem., January 19, 1996; 271(3): 1441 - 1447. [Abstract] [Full Text] [PDF]
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R. G. Boot, G. H. Renkema, A. Strijland, A. J. van Zonneveld, and J. M. F. G. Aerts Cloning of a cDNA Encoding Chitotriosidase, a Human Chitinase Produced by Macrophages J. Biol. Chem., November 3, 1995; 270(44): 26252 - 26256. [Abstract] [Full Text] [PDF]
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N. O. Keyhani, L.-X. Wang, Y. C. Lee, and S. Roseman The Chitin Disaccharide, N,N'-Diacetylchitobiose, Is Catabolized by Escherichia coli and Is Transported/Phosphorylated by the Phosphoenolpyruvate:Glycose Phosphotransferase System J. Biol. Chem., October 13, 2000; 275(42): 33084 - 33090. [Abstract] [Full Text] [PDF]
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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 Identification of a Novel Acidic Mammalian Chitinase Distinct from Chitotriosidase J. Biol. Chem., February 23, 2001; 276(9): 6770 - 6778. [Abstract] [Full Text] [PDF]
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Y.-J. Sun, N.-C. A. Chang, S.-I. Hung, A. C. Chang, C.-C. Chou, and C.-D. Hsiao The Crystal Structure of a Novel Mammalian Lectin, Ym1, Suggests a Saccharide Binding Site J. Biol. Chem., May 11, 2001; 276(20): 17507 - 17514. [Abstract] [Full Text] [PDF]
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N.-C. A. Chang, S.-I. Hung, K.-Y. Hwa, I. Kato, J.-E. Chen, C.-H. Liu, and A. C. Chang A Macrophage Protein, Ym1, Transiently Expressed during Inflammation Is a Novel Mammalian Lectin J. Biol. Chem., May 11, 2001; 276(20): 17497 - 17506. [Abstract] [Full Text] [PDF]
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