From the Institute of Microbiology and Immunology,
Institute of Neuroscience, and
§ Center for Neuroscience, School of Life Science, National
Yang-Ming University, Taipei, Taiwan 112, Republic of China, the
Institute of Biological Chemistry, Academia Sinica, Taipei,
Taiwan 115, Republic of China, and ** Biomedical Group, Takara Shuzo
Co., Ltd., Otsu, Shiga 520-21, Japan
Received for publication, November 16, 2000, and in revised form, January 16, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Oral infections of mice with Trichinella
spiralis induce activation of peritoneal exudate cells to
transiently express and secrete a crystallizable protein Ym1.
Purification of Ym1 to homogeneity was achieved. It is a single chain
polypeptide (45 kDa) with a strong tendency to crystallize at
its isoelectric point (pI 5.7). Co-expression of Ym1 with Mac-1 and
scavenger receptor pinpoints macrophages as its main producer. Protein
microsequencing data provide information required for full-length
cDNA cloning from libraries constructed from activated peritoneal
exudate cells. A single open reading frame of 398 amino acids with a
leader peptide (21 residues) typical of secretory protein was deduced
and later deposited in GenBankTM (accession number M94584)
in 1992. By means of surface plasmon resonance analyses, Ym1 has
been shown to exhibit binding specificity to saccharides with a
free amine group, such as GlcN, GalN, or GlcN polymers, but it
failed to bind to other saccharides. The interaction is
pH-dependent but Ca2+ and Mg2+
ion-independent. The binding avidity of Ym1 to GlcN oligosaccharides was enhanced by more than 1000-fold due to the clustering
effect. Specific binding of Ym1 to heparin suggests that
heparin/heparan sulfate may be its physiological ligand in
vivo during inflammation and/or tissue remodeling. Although it
shares ~30% homology with microbial chitinases, no chitinase
activity was found associated with Ym1. Genomic Southern blot analyses
suggest that Ym1 may represent a member of a novel lectin gene family.
Macrophages exhibit a myriad of critical functions in host defense
mechanism. The differentiation, activation, and effector functions of
macrophages have intrigued intensive studies (1-3). Macrophages are
responsible for antigen presentation and destruction against microbes
and neoplastic cells. In some instances, they serve as nonspecific
scavenger cells; in others, they may be subjected to modulation by
selective cytokines for enhanced competence to destroy facultative and
obligate intracellular parasites (4, 5). Furthermore, macrophages are
also producers of a wide variety of membrane and secretory proteins
pivotal for the development of tolerance, cell-cell recognition, and
cell-mediated cytotoxicity (6-8). As the list of known functions of
macrophages continues to grow, studies concerning the development of
competence to execute particular functions of this class of highly
versatile cells have brought consensus that macrophage activation is
enormously complex. It can be regulated by multiple signals, inductive
or suppressive, and by the sites of their residency and the biochemical
changes of the local milieu (9, 10).
In order to elicit emigrant peritoneal exudate cells
(PEC)1 as a source of
activated macrophages functionally distinct from that induced by
thioglycolate, Sephadex G-50, or Corynebacterium parvum, we
have adopted the paradigm of natural parasitic infection using
Trichinella spiralis, a systemic migration and
muscle-penetrating parasite (11, 12). We have previously noted that
marked cellular changes took place inside the peritoneal cavity of
T. spiralis-infected mice. Cellular accumulation peaks at
around day 15 postinfection (13). During this time, infective newborn
larvae will migrate via the peritoneal cavity of the host en route to
their final destination in the skeletal muscle cells (14).
The biochemical changes within the peritoneal cavity were also
monitored during the entire infection course. A novel protein transiently expressed by the lavaged PEC was identified (13). The
inducible expression of this protein and the profound cellular changes
paralleling its appearance suggested to us that it might bear
functional significance to the development of either host defense
against or tolerance to this nematode infection. We have designated
this protein as Ym1 and subsequently proceeded toward its purification
and molecular characterization (i.e. cloning, sequencing,
and expression). We report the following in the present study: (i) Ym1
is synthesized and secreted by activated macrophages during
inflammation elicited by parasitic infections; (ii) Ym1 is purified,
cloned, and characterized at both the protein and molecular levels;
(iii) Ym1 is a novel mammalian lectin exhibiting a
pH-dependent, specific affinity toward GlcN oligomers and
heparin, which may be structurally related to its natural ligand; and
(iv) Ym1 shares significant sequence homology with several mammalian chitinase-like proteins of unknown function reported recently. However,
none exhibits any chitinase activity, whereas our genomic Southern blot
analyses provide evidence supporting the notion that Ym1 and these
proteins may belong to a multigene family. The putative functional role
of Ym1 in vivo is discussed. In addition, the x-ray
structure of Ym1, the first of the gene family, is reported in the
accompanying article (79).
Induction of Ym1 Expression--
Infective larvae of T. spiralis were prepared from infected Sprague-Dawley rats as
described (15). Female ICR mice were orally infected with 250 (primary
infection) and 500 (secondary infection) T. spiralis larvae.
Groups of 3-6 mice were sacrificed at selected intervals
postinfection. PEC of both control and infected mice were
recovered by lavaging each with 5 ml of ice-cold phosphate-buffered saline (20 mM phosphate buffer, pH 7.4, 0.85% NaCl). Total
cell number in the lavaged fluid was enumerated and determined using a
hemocytometer. To purify activated macrophages, PEC (5 × 106 cells/75 mm2) were plated out in serum-free
Dulbecco's modified Eagle's medium supplemented with gentamycin (10 µg/ml), incubated in 5% CO2 at 37 °C for 2-3 h, and
washed three times with serum-free medium to remove nonadherent cells.
The percentage of attached cells capable of phagocytizing zymosan
particles estimated was routinely over 95% after differential Giemsa
staining. Both cell-free peritoneal exudate fluid (PEF) and cell
culture supernatant (PECCS) collected daily were used as sources
for Ym1 purification.
Purification of Ym1 by Preparative Polyacrylamide Gel
Electrophoresis (PAGE)--
For a typical run of purification, 50 ICR
mice were orally infected with 250 T. spiralis larvae. By
day 15, all animals were sacrificed to collect PEF and PECCS. Protein
concentrations were determined by Coomassie Blue assay reagent (Pierce)
before subjecting to preparative basic PAGE isolation according to
Davis (16). On the average, the amount of total proteins obtained from
PEF was 60 mg, and that from PECCS was 16 mg. However, we have
subsequently purified Ym1 using PECCS as starting materials, because
the composition of PECCS is much simpler than that of PEF. Basic PAGE
gel was packed into 18 tubes (0.6-cm inner diameter, 8-cm separating
gel, and 2-cm stacking gel). Approximately 0.9 mg of proteins were loaded on each gel. Gel discs containing Ym1 were pooled, diced into
small fragments, and dry-loaded onto the top of isoelectric focusing
(IEF) gels at the basic end. To protect the proteins, the gel fragments
were overlaid sequentially with 40, 20, and 10% sucrose solution
before filling up the tube with 0.4% ethanolamine. IEF-PAGE was
carried out according to O'Farrell (17) with the modification that no
urea was included in the gel. Broad range (pH 3.5-10, 18 tubes) and
narrow range (pH 5-7, 12 tubes) ampholytes (Amersham Pharmacia
Biotech) were used in two consecutive runs and loaded with 0.4 and 0.23 mg of proteins/tube, respectively. Gel segments containing visible Ym1
crystals were sliced out and rinsed with deionized and distilled water
to remove ampholytes. The purity and the relative molecular mass of Ym1
were determined by SDS-PAGE as described by Laemmli (18), with low
molecular weight standards (Amersham Pharmacia Biotech) included.
Production and Characterization of Ym1-specific
Antibodies--
Polyclonal antibodies against purified and
crystallized Ym1 were produced in rabbits. The specificity of the
antibodies was characterized by radioimmunoprecipitation (19) and
Western blots (20). For radioimmunoprecipitation, aliquots of
conditioned medium obtained from metabolically labeled
([35S]methionine, 20 µCi/ml, >1000 Ci/mmol;
PerkinElmer Life Sciences) PEC cultures were used. After incubation
with diluted (1:1000) primary antiserum or preimmune serum for 12-14
h, preabsorbed protein A-Sepharose 6B (50% suspension; Amersham
Pharmacia Biotech) was used to bring down the immune complex for
SDS-PAGE analyses in slabs using autoradiography (X-Omat films; Eastman
Kodak Co.). For Western analyses, samples separated in SDS-PAGE slab
gels were transferred onto Immobilon-P membrane (Millipore Corp.; 0.45 µm), and Ym1 proteins were detected using the
peroxidase-anti-peroxidase method of Sternberger et al.
(21). Cellular localization of Ym1 was examined in either a cytospin II
(Shandon) preparation of PEC obtained or cultured macrophages grown on
a chamber slide (Nunc) by the dual immunofluorescence test (22).
For cell type identification, the following antibodies were used:
R-phycoerythrin-conjugated anti-Mac-1 (M1/70.15; Cedarlane, Canada),
anti-scavenger receptor (2F8; Serotec), and anti-F4/80 (CI:A3-1;
Serotec). Dark field micrographs were taken in a Leitz microscope
(Ortholux II).
Amino Acid Composition and Protein Microsequencing--
Purified
and crystallized Ym1 was subjected to amino acid composition analyses
using a D400 amino acid analyzer (Durrum Co.). Amino acid sequences of
the NH2-terminal fragment and the CNBr fragments of
Ym1 were determined by protein microsequencing on an Applied Biosystems
1470 gas phase protein sequencer (23, 24).
Library Screening and DNA Sequencing--
Total RNA of PEC
collected from 50 mice orally infected with T. spiralis for
15 days were prepared by the guanidinium-CsCl centrifugation method
(25). Poly(A)+ mRNA purified by an oligo(dT) column
were primed by both oligo(dT) and random hexamers and custom
constructed into two cDNA libraries (CLONTECH);
PEC-1 cDNA library was packaged into a Southern Blot Hybridization--
Genomic DNA from testes of
129Sv mice was prepared as described (28). Aliquots of DNA (30 µg)
were digested with selected restriction enzymes as specified, separated
on 0.8% agarose gel, and transferred onto Immobilon-N membrane
(Millipore) before hybridization. Exon probes (Ep17-14 and Ep4-6) were
amplified from AC-11, and intron probe (Ip1-2) was amplified from a
3-kb EcoRI fragment of genomic DNA subcloned in
pBluescript-II SK Chitinase Assays--
Three different methods were used to
examine if purified, native Ym1 or recombinant Ym1 has chitinase
activity. The first method is based on an agar plate enzyme assay (29).
Briefly, 0.02% glycol chitin or glycol chitosan in an agar plate were
used as substrate. The second gel overlay method was carried out as described by Trudel and Asselin (30). Samples were resolved first by
10% basic native PAGE as described before. The third method was
performed with 2-aminopyridine-modified GlcNAc oligomers, GlcNAc3-6. The initial substrates and products were
separated on a PLAPAK type N column (Takara; 4.6 × 250 mm) and
detected by a fluorescent detector (excitation wavelength, 315 nm;
emission wavelength, 380 nm) on a high performance liquid
chromatography (HPLC) system (31).
Carbohydrate Binding Specificity Determined by Surface Plasmon
Resonance Technology--
BIAcore X (Amersham Pharmacia Biotech) was
used to screen for carbohydrate ligands that interact with Ym1. The
Biocore sensor chips CM5 and SA (streptavidin covalently
immobilized on a CM5 chip) and chemical activation reagents (amine
coupling kit) were obtained from Amersham Pharmacia Biotech. HBS-EP
buffer (10 mM HEPES with 150 mM NaCl, 3 mM EDTA, and 0.005% (v/v) surfactant P20) was used as a
continuous running buffer over the sensor surface in the BIAcore
experiments. Monosaccharides, various oligosaccharides, and
6-(biotinyl)-aminocaproylhydrazide (BACH) were from Sigma or Merck.
N-acetyllactosamine was from Calbiochem. Chitobiose (di-GlcN), chitotriose (tri-GlcN), and chitotetraose (tetra-GlcN) were
from Seikagaku.
In order to test the binding specificity of monosaccharides and GlcN
oligomers, purified Ym1 was immobilized onto the sensor chip CM5
surface via reaction with primary amines as suggested by the
manufacturer. Ym1 (100 µg/ml) in a 10 mM sodium acetate buffer (pH 4.25) or buffer control was introduced separately onto the
activated surface of flow cells number 1 and 2, respectively. Excess
N-hydroxysuccinimide ester groups were blocked by 1 M ethanolamine hydrochloride, pH 8.5, for 7 min.
Monosaccharides and GlcN oligomers in HBS buffer were injected across
the surface at a flow rate of 5-30 µl/min, and the real time binding
curves were observed. To stop the reaction, HBS buffer, pH 7.4, was
introduced onto the sensor chip to start the dissociation. The bulk
effect of refractive index changes was subtracted from in-line
reference flow cell to yield true binding response.
For calculation of KD and competition analyses,
biotinylated tetra-GlcN was immobilized onto a streptavidin chip (SA
chip). The biotinylation of tetra-GlcN with BACH was performed as
described (32). Briefly, GlcN oligomers (200 nmol) in 100 µl of water
were mixed with 100 µl of a 400-nmol solution of BACH in 30%
acetonitrile at 95 °C for 2 h. The product and initial substrates were analyzed by TLC. Approximately 40 nmol of GlcN oligomers was applied on HP-TLC Silica Gel 60 aluminum plates (Merck),
developed in n-propyl alcohol, water, 32% ammonia
(55:20:25, v/v/v). Biotinyl GlcN oligosaccharides, unlabeled GlcN
oligosaccharides, and BACH were visualized by spraying
The biotinylated tetra-GlcN (1 pmol) was reconstituted in HBS-P buffer
and immobilized onto an SA chip. Biotinyl tetra-GlcN (109 resonance
units) was captured in flow cell number 2. Flow cell number 1 on the
same sensor chip was used as a reference surface. Kinetic data were
calculated using the BIA evaluation software 3.0 (Pharmacia Biosensor
AB). The plot of subtracted responses versus the analyte
concentrations was used to derive the dissociation constant
(KD) in the Ym1-immobilized system. The
KD value of the tetra-GlcN immobilized assay system
was calculated by the simultaneous
ka/Kd fitting.
Heparin/Heparan Sulfate Binding Assay--
Various carbohydrates
or albumin/streptavidin-conjugated agarose beads were obtained from
Sigma and extensively washed with HBS-EP buffer. The biotinylated
di-GlcN was immobilized onto streptavidin-conjugated agarose beads. The
pH of T. spiralis-elicited PECCS containing Ym1 was first
adjusted to 4.75 with 50 mM sodium acetate buffer. To 100 µl of beads, 200 µl of conditioned medium was added. After a 2-h
incubation with gentle mixing at ambient temperature, the beads were
then extensively washed with HBS-EP buffer, pH 4.75, to remove the
nonspecific bound proteins. Proteins bound on the carbohydrate-conjugated beads were eluted in HBS-EP buffer, pH 7.4, and
the presence of Ym1 was revealed by Western blot.
Cellular and Biochemical Changes in the Peritoneal Cavity of T. spiralis-infected Mice--
Total cell counts in 5 ml of PEF recovered
from the lavages were plotted against time after infection. A
significant increase in the number of PEC at day 9 after primary
infection was noted as compared with control mice. The cell number
peaked at around day 15 (4.2 × 107 cells/mouse) and
subsided rapidly thereafter. The second infection of 500 T. spiralis larvae given at day 42 after primary infection elicited a
much faster and enhanced accumulation of PEC, which peaked at day 54 with total cell count (7.8 × 107 cells/mouse), almost
2 times higher than that of the primary response (Fig.
1A). The change of the protein
profile in PEF was monitored by basic PAGE as a function of time after
primary infection (Fig. 1B). Ym1 with a relative mobility
(Rm) of 0.77, was noted on the 15th day after
oral infection of T. spiralis. By day 18, the protein was no
longer detectable.
Purification of Ym1--
To purify the transiently expressed Ym1,
conditioned medium of cultured PEC (PECCS) of infected mice was
harvested, and a total of 16 mg of proteins were subjected to
preparative basic PAGE. Proteins in unstained gel slices with a
Rm of 0.77 were pooled, and ~7.4 mg of proteins
were subjected to IEF-PAGE in a broad pH gradient (pH 3.5-10). The gel
bands corresponding to pH 5.7 were again sliced out and pooled, and 2.8 mg of proteins were subjected to IEF-PAGE in a narrow pH gradient (pH
5-7) (Fig. 2A). After the
narrow IEF-PAGE, Ym1 actually is visible in gel as crystals without
staining (Fig. 2B). Under a phase-contrast microscope, Ym1
appears as cross- or scissors-like crystals (Fig. 2C). These
crystals may be eluted from the gel discs with 0.01 M
sodium bicarbonate buffer (pH 9.0) and recrystallized out after prolonged dialysis against deionized distilled water; a total of 2.3 mg
of Ym1 protein may be obtained. The crystals then acquire a square
sheet-like configuration and may "grow" into larger four-ridged pyramids (Fig. 2D). The purity of the protein band focused
at pI 5.7 was further verified by 10% SDS-PAGE. Results indicate that
Ym1 has been purified to homogeneity with an estimated molecular mass of 45 kDa (Fig. 2E). The final yield of purified and
crystallized Ym1 after dialysis was about 15% of the starting
materials.
Polyclonal Antibodies against Purified and Crystallized
Ym1--
After primary immunization, rabbits were subjected to at
least two additional booster injections. The specificity of antibodies was determined by Western blot of PECCS (Fig.
3A). An indirect immunofluorescence test was used to identify the cell type in PEC
responsible for Ym1 synthesis. To verify whether the Ym1-positive leukocytes are adherent cells, activated PEC were cultured with daily
medium change for 3 days to deplete nonadherent cells. Essentially all
adherent cells are Ym1-positive (Fig. 3B, a and
c), suggesting that activated peritoneal macrophages are
cells responsible for Ym1 expression. The notion was further supported
by co-localizing macrophage marker proteins Mac-1 (Fig. 3B,
b) and scavenger receptor (Fig. 3B,
d).
Molecular Cloning and Sequence Characterization--
Amino acid
composition analyses of Ym1 indicate the presence of ~34%
hydrophobic residues and relatively high content of glutamic acid
(9.1%) and aspartic acid (11.4%). The data are consistent with the
fact that Ym1 has a tendency to crystallize despite having an acidic pI
of 5.7. Protein microsequencing data of the NH2 terminus and three peptide fragments of Ym1 derived from CNBr cleavage revealed
a total of 100 residues. Pairs of primers were designed based on amino
acid sequences derived from NH2 terminus and two CNBr
fragments of purified Ym1. Two DNA fragments, Y1 (437 bp) and Y2 (605 bp) were initially amplified from PEC-1 cDNA library (
Homology search in the protein sequence data bank (GenBankTM/EBI
Data Bank) revealed that partial homology (11-36%) exists between Ym1
and members of the chitinase protein family. Chitinase activity of Ym1
was examined using a conventional agar plate assay, gel overlay assay,
and the more sensitive pyridylamination-HPLC method as described. No
chitinolytic activity was found associated with Ym1 either in the
purified form obtained from activated macrophages or in the recombinant
form expressed by the baculoviral expression system. Significant
homology (Fig. 5) has also been noticed
between Ym1 and several "chitinase-like" proteins reported
recently, namely human HC-gp39 (46% identity) (34), human
chitotriosidase (46%) (35), porcine gp38k (45%) (36), and
Drosophila DS-47 (25%) (37). With the exception of
chitotriosidase, it was intriguing to note that all of these proteins,
just as Ym1, do not exhibit chitinase activity.
Ym1 Presents as a Member of a Multiple Gene Family--
Genomic
Southern blot analyses were carried out using probes generated from
different parts of the coding region in order to determine whether Ym1
is encoded by a single copy gene and whether it contains introns.
Genomic DNA was subjected to restriction enzyme digestion before
hybridizing at high stringency to probe Ep17-14. Multiple bands of
different sizes were detected after digestion with EcoRI
(9.0, 5.3, and 3.2 kb), HindIII (4.6, 4.2, and 3.4 kb),
XbaI (8.0, 6.5, 3.2, and 2.0 kb), SacI (16 and
6.5 kb), and NcoI (15 and 2.0 kb), respectively (Fig.
6B). Data suggest the possible
existence of introns, pseudogenes, and/or other members of a gene
family. Detailed sequence studies of Ym1 phage clones purified from a
mouse (129Sv/J) genomic library revealed the consensus splice donor and
acceptor sites, defining at least 11 introns and 11 exons (Fig.
6A) in the coding region toward the 3'-untranslated region.
Reevaluation of the genomic Southern blot of Ym1 using intron-specific
probe Ip1-2 suggests that Ym1 is a single copy gene (Fig.
6C). Same pairs of exon and intron probes were employed in
murine chromosome mapping studies using the interspecific backcross analyses (in collaboration with Dr. N. G. Copeland, NCI-Frederick Cancer Research and Development Center). Results indicate that the
intron probe (Ip1-2) hybridized strongly to only one band in each
lane, while the exon probe hybridized fairly well to five bands in each
lane at relative low stringency. All of the bands hybridized with both
probes co-segregated and mapped to the middle region of mouse
chromosome 3. It thus appears that the two probes are recognizing a
family of closely related genes and that these genes are linked on
chromosome 3.2 Genomic
Southern blot analyses thereby suggest that Ym1 may represent a member
of a gene family.
Screen for Saccharides Interacting with Ym1--
Although Ym1 is
not a chitinase, significant sequence homology to chitinase would
suggest that Ym1 and other members of the "chitinase-like" proteins
may interact with saccharides sharing structural features similar to
chitin. We have therefore screened for carbohydrate ligands that will
interact with Ym1 bound on a CM5 sensor chip, using surface plasmon
resonance. As shown in Fig. 7, among
various monosaccharides tested, GlcN and GalN exhibited specific
binding to Ym1, whereas other monosaccharides tested did not. Data
obtained from oligosaccharides further indicated that those with free
amine groups are more potent ligands for Ym1 as compared with those
with N-acetylation.
Interaction of Ym1 and GlcN Oligosaccharides--
To optimize the
binding condition, the biotinylated tetra-GlcN was immobilized onto the
surface of a streptavidin chip, and the binding of soluble Ym1 was
recorded as a sensorgram. Data suggest that the binding is
pH-dependent; i.e. optimum binding occurs
between pH 4.5 to 5.0 (Fig.
8A). We have also found that the interaction did not require the presence of divalent cations (i.e. Ca2+ and Mg2+) (data not
shown). We have since conducted all assays at pH 4.75 in the absence of
divalent cations. To validate the binding specificity under acidic
assay conditions, GlcN oligomers were injected onto a Ym1-bound
surface at pH 4.75. The interaction appears in the form of square
waves, indicating that the interactions were fast. Similar to the
previous result (Fig. 7), the order of binding for GlcN oligomers at
steady state was tetra-GlcN > tri-GlcN > di-GlcN (Fig.
8B). Since the BIAcore has its limitation in measuring small analytes, direct measurement of the association and dissociation rates was difficult (38). Therefore, the affinity and kinetics of
binding between Ym1 and its carbohydrate ligands were determined with
the immobilized tetra-GlcN. The reaction showed relatively slow
association and dissociation (Fig. 8C). The on- and off-rate kinetics in tetra-GlcN immobilized assays were more amendable to
measurement. The dissociation rate constant (kd) was 7.5 × 10 Competition Analyses--
To verify the specificity of
binding by competition analyses, 250 mM monosaccharide
(GlcNAc, GalNAc, Glc, GlcN, or GalN) was incubated with Ym1
before co-injection onto the tetra-GlcN-bound chip. A significant
decrease in surface plasmon resonance was noted when GlcN or GalN was
used but not when GlcNAc, GalNAc, or Glc was used (data not shown).
Consistent with the data obtained from Ym1-immobilized assay (Fig. 7),
monosaccharides with the free amine groups such as GlcN and GalN are
capable of blocking the binding response, while similar monosaccharides
did not. To evaluate the clustering effect, GlcN oligomers at
concentrations as specified were incubated with Ym1 prior to injection
onto a tetra-GlcN-bound chip. Results obtained from the competition
analyses indicate that all GlcN saccharides tested were effective
competitors; however, the potency increases as a function of valences
(Fig. 8D). The IC50 was determined to be ~100
mM for GlcN, <500 µM for di- and tri-GlcN,
and 100 µM for tetra-GlcN, respectively. The fact that
tetra-GlcN is 1000-fold more effective than GlcN monomer suggests that
Ym1 is a mammalian lectin that exhibits specific binding toward
multivalent hexosamines, and a clustering effect was observed in the
interaction between Ym1 and GlcN oligomers.
Binding Property of Ym1 to Heparin/Heparan Sulfate--
Ym1 was
also examined for its ability to bind to different carbohydrate ligands
by affinity chromatography and Western blot. Among all ligands tested,
only di-GlcN and heparin demonstrated the ability to retain Ym1 as
evidenced in the Western blots. Albumin and streptavidin were included
as negative control (Fig. 9). The relative amount of Ym1 bound by di-GlcN is apparently less in contrast
to that bound by heparin. Variations in intensity of Ym1 retained among
three types of heparin used might reflect the amount of heparin
conjugated onto the agarose beads. The pH of the buffer used to
dissociate Ym1 from carbohydrate-conjugated beads was critical, since
the specific binding to heparin was also pH-dependent. In
other words, optimum binding occurs at pH 4.75, whereas to elute Ym1
from heparin-agarose beads, HBS-EP buffer at pH 7.4 is more effective
than acidic pH.
Macrophages are perhaps one of the most versatile cell types in
the body, participating in a vast array of biological processes from
fighting infections to tissue remodeling and wound healing. The
diversity of its functional repertoire strongly suggests that its
differentiation and activation may be subjected to the profound influence of environmental changes (39-42). We have adopted a paradigm of natural parasitic infection using T. spiralis in order to
further explore the roles of activated macrophages during the course of a complex immune response evoked by the host against parasites. Cellular infiltration in the peritoneal cavity peaked around day 15 and
day 12 after the primary and secondary infection, respectively (Fig.
1A). The shorter time course required for cell accumulation after the secondary infection reflects a typical memory response. Transient appearance of Ym1 paralleling the rise and rapid fall of the PEC numbers during the course of inflammation was observed. The
peak of Ym1 expression occurs at a stage during infection right after
the intestinal expulsion of the adult worms and at the beginning of an
intense systemic migration of newborn larvae throughout the body of the
host (13). The peritoneal cavity at this time would have experienced a
traumatic process that calls for tissue repair. The transient
appearance of Ym1 (Fig. 1B) would suggest that it may be
involved in the establishment of an inflammatory management control in
the peritoneal cavity. To establish its identity as an immune mediator
and its cellular origin, purification and molecular characterization of
Ym1 were essential. We have achieved its purification by preparative
polyacrylamide gel electrophoresis. During the process, it was rather
surprising to note that Ym1 would invariably be focused into a visible
white, sandy-like band in the IEF-PAGE gel (Fig. 2B). Under
a phase-contrast microscope, the sandy precipitates appear in the form
of either a cross or a scissors-like configuration (Fig.
2C). After elution and removal of ampholytes, crystals in
square sheets or pyramids were clearly visible (Fig. 2D).
Crystallized Ym1 was indeed the purified and homogeneous preparation of
Ym1 as evidenced by data obtained from SDS-PAGE. Ym1 has an estimated
molecular mass of 45 kDa and an isoelectric point of 5.7 (Fig.
2E). The strong tendency of Ym1 to form crystals is further
supported by the recent report that a crystal form of Ym1 may be
found in the lungs of mutant mice with immunodeficiency (43). We have
then taken advantage of Ym1 being a crystallizable protein to achieve
its structure analyses by x-ray crystallography (79).
Specific polyclonal antibodies were raised against purified Ym1.
Immunofluorescence staining revealed that in smear or cytospin preparations, only subpopulations of PEC were Ym1-positive. However, in
cultured preparations, after daily change of medium for 3 days to
remove all nonadherent cells (i.e. lymphocytes), the
majority of adherent cells (>90%) were Ym1-positive (Fig. 3). Data
thereby suggest that adherent macrophage is probably the cell type
responsible for Ym1 synthesis and secretion. The notion was further
supported by co-localization of macrophage marker proteins (44, 45), scavenger receptor and Mac-1 with Ym1 in the same cell (Fig. 3). In
addition, when macrophages harvested 15 days post-T.
spiralis infection were metabolically labeled with a
14C-labeled amino acid mixture and subjected to IEF-PAGE,
labeled crystals of Ym1 were revealed by autoradiography (data not
shown). This result further supports the possibility that Ym1 is
de novo synthesized and secreted by adherent macrophages.
The kinetics of Ym1 secretion has also been studied by metabolic
labeling techniques. Cultured macrophages were first starved in
methionine-free Dulbecco's modified Eagle's medium for 1 h,
pulsed with [35S]methionine for 10 min, and chased from 2 to 20 h in medium containing 0.1 mM nonradioactive
L-methionine. The secretion of newly synthesized and
thereby labeled Ym1 was monitored as a function of time by radioimmunoprecipitation. The results indicated a half-maximal release
time of 4-6 h for Ym1. Upon the addition of the Ca2+
ionophore A23187, 10 nM was sufficient to evoke a much
enhanced secretion of newly synthesized Ym1 presumably due to
Ca2+ influx (data not shown).
The developmental and spatial expression pattern of Ym1 in normal or
infected mice has been examined by Western blot and immunocytochemical analyses. Constitutive, basal expression of Ym1 is restricted to bone
marrow, spleen, and lung, whereas transient or inducible expression may
be detected in embryonic liver and at additional sites (e.g.
peritoneal cavity and brain), where inflammation or tissue injury was
introduced.3 These
results are consistent with the fact that fetal liver and bone marrow
are the original sources of myeloid lineage cells, which will
eventually develop into tissue macrophages (46, 47). In our experience,
Ascaris suum was also able to induce the production of Ym1
in the peritoneal cavity to a comparable level. However, agents such as
thioglycolate, Sephadex G-50, and C. parvum could elicit the
accumulation of active peritoneal macrophages 3-4 days after
peritoneal injections to a much lesser extent. None of these agents
would induce the expression of Ym1 in quantity that permitted its
purification (data not shown).
To delineate the function of Ym1, we proceeded toward its gene cloning.
Molecular cloning of Ym1 cDNA was achieved by PCR employing
biochemical data obtained from microsequencing of the purified and
crystallized protein. Amino acid sequence deduced from the
cDNA revealed the existence of a typical Kozak motif, and a
signal peptide. In addition, sequences corresponding to the
NH2-terminal fragment of Ym1 (secretory form) and that of three CNBr cleavage peptides are also present (Fig. 4). The calculated pI of 5.4 and molecular mass of 45 kDa are also consistent with the
biochemical features of the purified Ym1 determined empirically. Furthermore, cDNA-directed synthesis and secretion of recombinant Ym1 in both mammalian and baculoviral expression systems were validated
using specific antibodies in Western blot analyses (data not shown). We
have thereby concluded that the cDNA clones on hand do contain the
complete sequence information for encoding the secretory Ym1, a protein
transiently expressed by activated macrophages during the course of
inflammation against parasitic infections. We were the first to submit
the Ym1 cDNA and protein sequences to GenBankTM. An
accession number of M94584 was assigned to Ym1 in 1992. Recently,
Owhashi et al. (48) reported the cloning of a cDNA (submitted in 1996, GenBankTM accession number D87757) that
encodes a protein with a sequence identical to Ym1. The protein was
accredited with a function as eosinophil chemotactic cytokine (ECF-L)
(48). We are conservative about this functional assignment for the
following reasons. (i) Chemotactic activity was one of the initial
activities we examined when purified and crystallized Ym1 was
available. No chemotactic activities were ever found associated with
purified Ym1 in either in vitro chemotaxis assays or
in vivo assessment by intradermal injections on the back of
mice. (ii) Ym1 is expressed by cells of myeloid lineage such as
activated macrophages and myeloid progenitors in bone marrow. It has
never been detected in the lymphoid system, e.g. thymus and
lymph node.3 In addition, selective up-regulation
of Ym1 has been reported in a committed myeloid progenitor cell line
EPRO but not in a multipotent cell line EML capable of differentiating
into lymphoid cells (49). (iii) In their own report, Owhashi et
al. (48) stated that the calculated isoelectric point (pI) derived
from cDNA-deduced protein is 5.3, whereas pI of the
chemotactic activity was determined empirically to be 3.6. A difference
of 1.7 in pH units would strongly imply that the amino acid composition
of the protein with the chemotactic activity is different from that of
the protein whose cDNA they have cloned. (iv) In their
assays, the chemotactic activity expressed by the postinfection
splenocytes (or CD8+ T-cells) was dependent on the
inclusion in the assay of parasitic extracts (i.e. 5-15
µg/ml Toxocara canis (50) or 100 µg/ml
Mesocestoides corti larvae (48)); both are mixtures of
unknown compositions and activities.
Molecular cloning of the Ym1 gene from a mouse genomic library (129 Sv/J) was pursued in parallel for the establishment of knock-out mice.
Genomic organization of the Ym1 gene revealed the existence of at least
11 exons (Fig. 6A). The complex pattern derived from the
exon probe and the rather simple pattern derived from the intron probe
(Fig. 6B) suggest the possible existence of pseudogenes or a
family of closely related genes. Using the same pairs of probes, murine
chromosome mapping studies revealed that the intron probe (Ip1-2)
hybridized strongly to only one band in each lane, while the exon probe
hybridized well to five bands in each lane at relatively low
stringency. In addition, bands hybridized with both probes
co-segregated and mapped to the middle region of mouse chromosome 3. Multiple genes hybridized by exon probes are not pseudogenes but more
likely different members of a closely related gene family. Employing
our Ym1 cDNA sequence as probes, a separate study reported recently
by Jin et al. (51) has reached the same conclusion.
Approximately 30% homology was revealed between Ym1 and many members
of the microbial chitinase gene family (52, 53). However, no
chitinolytic activity was detected in either purified or recombinant
Ym1 using three different assay methods as stated under "Materials
and Methods." The notion that Ym1 is not a chitinase was further
supported by the fact that the two conserved acidic residues key to
chitinase activity (e.g. Asp200 and
Glu204 of Bacillus enzyme) are Asn and Gln in
Ym1, respectively. Site-directed mutagenesis studies of Watanabe
et al. (54) have clearly demonstrated that mutations from
Asp200 to Asn200 or from Glu204 to
Gln204 will both severely impair the enzyme activity. In
addition to microbial chitinases, significant homology was also found
between Ym1 and several "chitinase-like" proteins reported recently
(i.e. human cartilage HC-gp39, human macrophage
chitotriosidase, porcine smooth muscle gp38k, and Drosophila
DS-47) (Fig. 5). Human HC-gp39 was first reported as a secretory
glycoprotein found in the inflamed joint of arthritis patients only
(34). The cell type responsible for HC-gp39 production was subsequently
verified as the differentiating macrophages (55, 56). Human macrophage
chitotriosidase is a chitinolytic enzyme found markedly elevated in
plasma of Gaucher disease patients (57, 58). Porcine gp38k is a
secretory glycoprotein synthesized by vascular smooth muscle cells
during differentiation from monolayer to nodular form (59).
Drosophila DS-47 is synthesized and secreted by
macrophage-like hemocytes and cells in the fat body, which is
equivalent to mammalian liver (37). Several intriguing points have
emerged after comparing features unique to Ym1 and these proteins. (i)
Just like Ym1, all share sequence similarity with chitinase, but none
exhibits the respective enzymatic activity, with the exception of
chitotriosidase. However, it is possible that their substrates may
share structural features similar to chitin. (ii) All are secretory
proteins, suggesting that their sites of action are most likely to be
extracellular. (iii) All are inducible; peak expression is found during
inflammation, development/differentiation, tissue remodeling, and/or
wound healing. (iv) Despite all similarities, tissue-specific
expression patterns of these proteins would argue against their being
species variants of the same protein. It is conceivable that this group
of proteins of "unknown" functions (except chitotriosidase) may be
members of the Ym1 gene family, especially when one
considers that HC-gp39 and chitotriosidase were mapped to the same
chromosome (chromosome 1) where the human homologue of Ym1 and its gene
members would be located (34, 51, 60).
Parallel studies of Ym1 using x-ray crystallography (79) revealed that
Ym1 has a In addition, the clustering effect, a characteristic feature in
carbohydrate-lectin interaction (65, 66), was revealed in the
competition analyses, in which inhibition of binding by tetra-GlcN is
1000-fold more effective than that by monomers of GlcN (Fig.
8D). Although numbers of macrophage-derived lectins have
been identified and reported with binding specificity to mannose,
mannose 6-phosphate, or Gal/GalNAc, respectively (67-69), Ym1, a
secretory mediator produced by activated macrophages during inflammatory response, is a novel lectin with a GlcN binding
specificity. To identify other ligands of Ym1, its ability to interact
with heparin was examined. Specific binding of Ym1 to heparin in
addition to GlcN dimers was revealed (Fig. 9). Although it is not clear how Ym1 interacts with heparin and if Ym1 interacts with heparan sulfate, we postulated that Ym1 might interact with heparin/heparan sulfate proteoglycans (HSPGs) via GlcN, as selectins (70). Further analysis should be performed to address this issue.
HSPGs have been recognized as ubiquitous ligands on cell surface and in
extracellular matrix (ECM) (71). Enormous structural heterogeneity can
be generated through specific HSPG chain modifications during their
biosythesis, as well as from the diverse nature of their core proteins.
The GlcN residues in HSPGs may be N-sulfated, N-acetylated, or N-unsubstituted. Proteins
anchored on glycosaminoglycan side chains of HSPGs may serve a variety
of functional purposes, from simple immobilization or protection
against degradation to modulation of distinct biological activities
(72, 73). Induced expression of HSPGs (i.e. syndecan-2) on
the surface of activated human macrophages has been reported (74). The
functional significance of this transient and selective expression of
HSPGs was elucidated as to deliver the sequestered growth factors
(e.g. fibroblast growth factor, vascular endothelial growth
factor, epidermal growth factor) also produced by inflammatory
macrophages, to their appropriate receptors on fibroblasts or
endothelial cells for signaling new tissue growth during the repair
processes (74). Syndecan-1 has been identified as the major
HSPG on murine macrophage cell surface (75). Whether secretory
Ym1 would be sequestered by binding to syndecan-1 and delivered by its
producing macrophages to its site of action warrants further study.
Alternatively, inflammatory responses elicited by tissue injury or
infections require the emigration of subsets of leukocytes from
circulation to the inflamed loci. Selective recognition between
extravasated leukocytes, endothelial cells, and the local ECM adjacent
to the inflammatory tissues all involve lectin-carbohydrate binding
(76, 77). Evidence supports the possibility that the nature of a
particular inflammatory stimulus determines the extravasation of
certain immune cell subtypes and the compositional changes of ECM. The
immune mediators secreted by the leukocytes would act in concert with
ECM to either promote or diminish the inflammatory responses (42).
Reports have established that the N-unsubstituted GlcN
residues are enriched in native heparan sulfate species capable of
binding to P- and L-selectins and thus potentially involved in
regulating leukocyte traffic (70, 72, 78). Based on the observations in
the present study, secretory Ym1 is transiently expressed in the
peritoneal cavity after T. spiralis infection. Its inducible
expression is not at the initial stage of infection but rather at a
stage (day 15-17) when the infiltrating leukocytes are
"leaving" the inflammatory peritoneal cavity, as evidenced by the
parallel rapid fall of total cell counts (Fig. 1A). With a
binding specificity similar to that of the homing receptors (selectins)
of leukocytes, it is tempting to propose that a pulse of Ym1 secretion
at the inflammatory foci was competing for binding sites on local ECM
occupied by the infiltrating leukocytes. Diminished local inflammatory
reactions may subsequently initiate the reestablishment of homeostasis. Structure features of Ym1 responsible for GlcN binding are reported in
the accompanying x-ray crystallography study (79), which will provide
definitive insights for future design of agents that may be important
for prevention and/or treatment of inflammation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
gt11 vector, whereas
PEC-2 cDNA library was packed into a
ZAP-II vector. In order to
prepare Ym1-specific probes by polymerase chain reaction (PCR) (26), a
sense primer was designed based on the sequence of the NH2
terminus (P1,
5'-ATGTG(T/C)TA(T/C)TA(T/C)ACICA(T/C)TGGGCIAA(A/G)GA-3'). Antisense primers were designed based on sequences derived from CNBr
fragment 2 (P2, 5'-TT(T/C)TCICCIGT(A/G)TAICC(A/G)TC(T/C)TT-3') and 3 (P3, 5'-TC(T/C)TC(T/C)TC(A/G)AAIGC(T/C)TTIC(G/T)CAT-3'), respectively.
PEC-1 cDNA library was screened by PCR using primer pairs as
designed. PEC-2 cDNA library and a mouse lung cDNA library (Stratagene,
ZAP-II vector) were also screened using partial clones
of Ym1; PEC21 and PEC15 were obtained from PEC-1 library as probes.
Full-length cDNA clones (AC-11 and lung clone 6-1) containing the
complete coding region of Ym1 were later identified and purified from
both libraries, respectively. Nucleotide sequence was determined
according to the dideoxy chain termination method of Sanger et
al. (27). Mouse genomic library 129Sv in
FIXII (Stratagene) was
screened using the same probes in search of genomic clones of Ym1. A
total of 107 phage clones were screened, and 15 clones were
amplified, purified, and sequenced.
(Stratagene) by PCR, respectively.
Probe labeling, prehybridization, and hybridization were conducted as
described (28). Primer pairs used were as follows: for Ep17-14, Ep17
(5'-TGGAAGGACCATGGAGCAGC-3') and Ep14 (5'-GCCTTCAACTTGAAGCTCC-3'); for
Ep4-6, Ep4 (5'-CATCTCTTCAGTGTTCTGG-3') and Ep6
(5'-AGACCTCAGTGGCTCCTTCA-3'); for Ip1-2, Ip1
(5'-GTTATCATCTACCACTCC-3') and Ip2 (5'-ATGTTGGTTCCATGTTGGG-3').
-naphathol/H2SO4 onto a reference lane, and
the desired product was identified and scraped from the plate. The
purified biotinylated GlcN oligomers were extracted with
n-propyl alcohol, water, 32% ammonia (55/20/25, v/v/v)
twice. The purity of extracted biotinylated GlcN oligomers was
confirmed by HP-TLC and the molecular weight of purified biotinylated tetra-GlcN was identified by FAB-mass spectrometry at a positive ion
mode and revealed abundant ions at m/z 1038 (MNa+) and m/z 1016 (MH+).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (90K):
[in a new window]
Fig. 1.
Cellular and biochemical changes in
peritoneal cavity of T. spiralis-infected mice.
PEC and PEF were harvested from T. spiralis infected mice at
3-day intervals. A, the total number of PEC in 5 ml of
lavaged fluid of each time point was enumerated and plotted as a
function of time postinfection: primary at day 0 and secondary at day
42. The cell counts peaked at around day 15 and day 12 after the
infections, respectively. The number of cells infiltrated to the
peritoneal cavity after the secondary infections is ~2-fold higher.
B, the protein profile of PEF was resolved on basic
PAGE as a function of time postprimary infection. The arrow
denotes the appearance of Ym1 at day 15.
View larger version (147K):
[in a new window]
Fig. 2.
Purification and crystallization of Ym1.
A, Coomassie Blue staining pattern of Ym1 revealed through a
series of gel electrophoresis. Lane 1, the
bracket denotes the position of Ym1 after PECCS was resolved
on basic PAGE. Lanes 2 and 3, the
arrows denote Ym1 after broad range (pH 3.5-10) and narrow
range (pH 5-7) IEF-PAGE, respectively. B, Ym1 crystals of
sandy appearance may be visualized in gel discs collected after
isoelectric focusing gel electrophoresis. C, cross- or
scissors-like Ym1 crystals in gel visualized under a stereomicroscope.
D, after extensive dialysis, the purified Ym1 formed
crystals in shapes such as a square sheet (arrow) or
pyramid. E, the purity of Ym1 was evaluated (lane
5), and the molecular mass (arrow) was determined
(lane 4) in SDS-PAGE.
View larger version (34K):
[in a new window]
Fig. 3.
Specificity of polyclonal antibodies against
Ym1 and subcellular co-localization of macrophage protein markers with
Ym1. A, proteins of PECCS were resolved by SDS-PAGE,
either stained with Coomassie Blue (lane
1) or subjected to Western blot, to validate the specificity
of Ym1 antibodies (lane 2) directed against
purified and crystallized Ym1 versus the preimmune
serum (lane 3). B, PEC harvested 15 days after T. spiralis infection were cultured and
double-labeled with specific antibodies against Ym1 (a
and c) and macrophage markers Mac-1 (b) or
scavenger receptor (d) respectively (original magnification, × 400).
gt11) by
PCR. DNA sequence data obtained indicated that Y1 and Y2 are
overlapping clones and contain partial sequences identical to that
derived from protein microsequencing of Ym1 (Fig.
4). After screening the PEC-1 cDNA
library with Y1 and Y2, Ym1-positive clones were identified, purified,
subcloned, and sequenced. Two clones (PEC21 (1035 bp) and PEC15 (1475 bp)) overlapping by as many as 970 bp, constitute a single open
reading frame of 398 amino acids. The same sequence was enclosed in a
single cDNA clone (AC-11) after rescreening a second PEC cDNA
library (PEC-2,
ZAP-II) (Fig. 4). In addition, a typical eukaryotic
ribosome binding motif (33) adjacent the putative initiator codon is
present. The first 21 amino acids also fit well as the predicted
secretory signal peptide with a cleavage site at residue Y, which is
the NH2-terminal residue of the purified Ym1. Sequences of
all peptide fragments obtained from protein microsequencing analyses
are present in the deduced open reading frame of Ym1 (Fig. 4). The
calculated pI value (5.4), molecular weight (44,456), and amino acid
composition using PC Gene (IntelleGenetics) correlate well with the
empirical results obtained from the purified Ym1. The DNA and the
deduced protein sequence of Ym1 were submitted to GenBankTM
in 1992 and assigned with accession number M94584.
View larger version (63K):
[in a new window]
Fig. 4.
Full-length cDNA sequence and the deduced
amino acid sequence of Ym1. A single PEC cDNA clone (1526 bp)
encoding the full-length Ym1 was identified and sequenced. The open
reading frame consists of a protein of 398 amino acids with a typical
Kozak sequence (underlined) adjacent the ATG start codon, a
putative signal peptide of 21 amino acids (in boldface
type), and a stop codon mapped at nucleotide position 1195. The
amino acid sequence deduced from the cDNA as the secretory form of
Ym1 started at residue 22, and the peptide sequence (boxed
from residue 22 to 48) is in complete agreement as the NH2
terminus sequence derived from peptide microsequencing of purified Ym1.
Boxed sequences of residues 109-134, 162-192, and 210-225
are also identical to those derived from the CNBr peptides of
purified Ym1. Corresponding nucleotide sequences that are
shaded denote the primer pairs designed for generating PCR
products as specific probes of Ym1.
View larger version (90K):
[in a new window]
Fig. 5.
Multiple sequence alignment of Ym1 with
members of the chitinase-like protein family. The amino acid
sequences of mouse Ym1 (M94584), human HC-gp39 (M80927), human
chitotriosidase CHIT1 (U29615), porcine gp38k (U19900),
Drosophila DS-47 (U13825), and chitinase A1 of
Bacillus circulans ChiA1 (P20533) are shown. Those residues
in total identity for all six members are marked in pink;
those that are identical in 3-5 members are marked in
yellow, and those with similar properties to the
yellow blocks are marked in green. N
and Q marked in blue denote the positions of
residues key to chitinase activity.
View larger version (65K):
[in a new window]
Fig. 6.
Southern blot analysis of mouse genomic
DNA. A, the genomic organization of the Ym1 gene is
depicted in the schematic diagram, where at least 11 exons may be
identified in cDNA. The thick bars mark the
location of exon probes (Ep17-14 and Ep4-6) and intron probe (Ip1-2).
Mouse genomic DNA (30 µg) digested with EcoRI,
HindIII, XbaI, SacI, and
NcoI were hybridized with labeled exon probe, Ep17-14
(B), or intron probe (C) derived from PCR. The
positions of size markers are marked. The asterisks denote
the possible corresponding exon fragments.
View larger version (12K):
[in a new window]
Fig. 7.
Screening of saccharides interacting
with surface-bound Ym1. Purified Ym1 was covalently immobilized
onto the surface of sensor chip (3679 resonance units
(RU)/CM5 chip). Saccharides in HBS-EP buffer (pH 7.4) were
injected separately at a flow rate of 5 µl/min. Interaction of bound
Ym1 with various saccharides at concentrations specified was
assessed via surface plasmon resonance analysis and plotted as
relative response at the equilibrium by subtracting the response from
the reference cell. Man, mannose; Fuc,
L-( )-fucose; Rha,
-L-rhamnose;
Fru,
-D-(
)-fructose;
Xyl, D-(+)-xylose; Suc, sucrose;
Mal, maltose; LacNAc,
N-acetyllactosamine.
4 s
1,
with an association rate constant (ka) of 3.36 × 103 M
1
s
1. The KD was then
derived from kd/ka to be 223 nM.
View larger version (24K):
[in a new window]
Fig. 8.
Characterization of interaction between Ym1
and GlcN oligosaccharides. A, the effect of pH on Ym1
binding to immobilized tetra-GlcN (109 resonance units
(RU)/SA chip) was examined. Soluble Ym1 (2.8 µM) in buffer with pH as specified was injected and
allowed to interact with the tetra-GlcN-coated surface. The responses
were measured across a broad pH range from 4.0-9.0. Optimal binding
was observed at a narrow pH range from 4.5 to 5.0. B, the
relative binding affinity of GlcN oligosaccharides to immobilized Ym1
on a CM5 chip at pH 4.75 was examined. GlcN oligomers (0.8 mM) were separately injected over the Ym1-immobilized
surface, and the sensorgrams were monitored at a flow rate of 30 µl/min for 180 s. C, binding kinetics of Ym1 to
immobilized tetra-GlcN at pH 4.75 were examined. The reaction is
concentration-dependent, and the slower kinetics in this
binding paradigm allowed the estimation of the KD.
D, competition assays with GlcN saccharides. The binding of
Ym1 (1.4 µM) to immobilized tetra-GlcN at pH 4.75 was
competed by co-injection of various GlcN saccharides at specified
concentrations. , GlcN monomer;
, di-GlcN; ×, tri-GlcN;
,
tetra-GlcN. The ability of GlcN saccharides to compete for Ym1 was
plotted as percentage inhibition.
View larger version (11K):
[in a new window]
Fig. 9.
Identification of heparin/heparan sulfate as
ligands of Ym1. PECCS, the conditioned medium derived from
T. spiralis-elicited peritoneal exudate cells was incubated
with agarose beads conjugated with different carbohydrates as
specified. Conjugated moieties capable of binding to Ym1 were
identified in Western blot by specific antibodies against Ym1. PECCS
containing Ym1 was included as a positive control, whereas
streptavidin- and albumin-conjugated beads were included as negative
controls.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
TIM barrel structure shared by many glycolytic
enzymes and proteins that bind and transport metabolites (61). This
further supports the notion that Ym1 may interact with its natural
ligand bearing carbohydrate moiety. The binding properties of Ym1 to
various saccharides were evaluated by a biosensor based on surface
plasmon resonance (BIAcore) (62, 63). Data indicate that Ym1 binds not
to chitin (oligomer of GlcNAc) but to saccharides with a free amine
group, such as GlcN, and its oligomers (Fig. 7). To increase
the sensitivity of detection, biotinylated tetra-GlcN was immobilized
onto the sensor chip for all subsequent binding studies (64). The
optimum pH for the binding is in the range of pH 4.5-5.0 (Fig.
8A), which would suggest that the electrostatic force
between Ym1 and GlcN saccharides is important, since the binding was
significantly affected by pH. The affinity between Ym1 and tetra-GlcN
was determined to be about 223 nm.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. John M. Gardnar (Protein Structure Laboratory at the University of California, Davis) for the peptide microsequencing work and Takara Shuzo Co. Ltd. (Osaka Japan) for assaying the chitinase activity using the PA method. Professor Albert M. Wu's valuable suggestions on carbohydrate binding studies are greatly appreciated. Weber Chern is acknowledged for excellent help for all the graphic illustrations.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the grants provided by the National Science Council of the Republic of China.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.
This paper is dedicated to Dr. William Zimmermann, a memorable mentor to N. C. Chang, for support and encouragement during the initial pursuit of this work.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M94584.
The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI accession number AAB62394.
¶ To whom correspondence should be addressed: Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei, Taiwan 112, Republic of China. Tel.: 886-2-2826-7114; Fax: 886-2-2820-2593; E-mail: acchang@ym.edu.tw.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M010417200
2 Chromosomal mapping by N. G. Copeland and N. C. Chang, unpublished results.
3 S. I. Hung, A. C. Chang, I. Kato, and N. C. Chang, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PEC, peritoneal exudate cell(s); PEF, peritoneal exudate fluid; PECCS, PEC culture supernatant; PAGE, polyacrylamide gel electrophoresis; IEF, isoelectric focusing; PCR, polymerase chain reaction; HPLC, high performance liquid chromatography; HSPGs, heparin/heparan sulfate proteoglycan(s); ECM, extracellular matrix; BACH, 6-(biotinyl)-aminocaproylhydrazide; bp, base pairs; kb, kilobase pair(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Gordon, S., Clarke, S., Greaves, D., and Doyle, A. (1995) Curr. Opin. Immunol. 7, 24-33[CrossRef][Medline] [Order article via Infotrieve] |
2. | MacMicking, J., Xie, Q. W., and Nathan, C. (1997) Annu. Rev. Immunol. 15, 323-350[CrossRef][Medline] [Order article via Infotrieve] |
3. | Aderem, A., and Underhill, D. M. (1999) Annu. Rev. Immunol. 17, 593-623[CrossRef][Medline] [Order article via Infotrieve] |
4. | Bogdan, C., and Nathan, C. (1993) Ann. N. Y. Acad. Sci. 685, 713-739[Abstract] |
5. | Mosser, D. M., and Karp, C. L. (1999) Curr. Opin. Immunol. 11, 406-411[CrossRef][Medline] [Order article via Infotrieve] |
6. | Gregory, C. D., Devitt, A., and Moffatt, O. (1998) Biochem. Soc. Trans. 26, 644-649[Medline] [Order article via Infotrieve] |
7. | Nathan, C. F. (1987) J. Clin. Invest. 79, 319-326[Medline] [Order article via Infotrieve] |
8. |
Zeisberger, E.,
and Roth, J.
(1998)
Ann. N. Y. Acad. Sci.
856,
116-131 |
9. | Russell, D. G. (1995) Curr. Opin. Immunol. 7, 479-484[CrossRef][Medline] [Order article via Infotrieve] |
10. | Gordon, S. (1998) Res. Immunol. 149, 685-688[CrossRef][Medline] [Order article via Infotrieve] |
11. | Wakelin, D. (1993) J. Parasitol. 79, 488-494[Medline] [Order article via Infotrieve] |
12. | Bell, R. G. (1998) Adv. Parasitol. 41, 149-217[Medline] [Order article via Infotrieve] |
13. | Chang, N. C., Chang, J. Y., and Zimmermann, W. L. (1982) Fed. Proc. 41, 561 |
14. | Despommier, D. D. (1993) J. Parasitol. 79, 472-482[Medline] [Order article via Infotrieve] |
15. | Chang, N. C., Dellmann, H. D., Kjaersgaard, P., Jeska, E. L., and Zimmermann, W. L. (1989) in Trichinellosis (Tanner, C. E. , Martinez-Fernandez, A. R. , and Bolas-Fernandez, F., eds) , pp. 304-311, Consejo Superior de Investigacìones Cientificas Press, Madrid, Spain |
16. | Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404-427[Medline] [Order article via Infotrieve] |
17. | O'Farrell, P. H. (1975) J. Biol. Chem. 250, 4007-4021[Abstract] |
18. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
19. | Goding, J. (1983) in Monoclonal Antibodies: Principles and Practice (Goding, J., ed) , pp. 134-187, Academic Press, London |
20. | Towbin, H., and Gordon, J. (1984) J. Immunol. Methods 72, 313-340[CrossRef][Medline] [Order article via Infotrieve] |
21. | Sternberger, L. A., Hardy, P. H., Jr., Cuculis, J. J., and Meyer, H. G. (1970) J. Histochem. Cytochem. 18, 315-333[Medline] [Order article via Infotrieve] |
22. | Johnstone, A., and Thorpe, R. (1987) in Immunocytochemistry in Practice (Johnstone, A. , and Thorpe, R., eds), 2nd Ed. , pp. 261-289, Blackwell Scientific Publications, Oxford |
23. |
Matsudaira, P.
(1987)
J. Biol. Chem.
262,
10035-10038 |
24. |
Moos, M., Jr.,
Nguyen, N. Y.,
and Liu, T. Y.
(1988)
J. Biol. Chem.
263,
6005-6008 |
25. | Berger, S. L. (1987) Methods Enzymol. 152, 215-219[Medline] [Order article via Infotrieve] |
26. | Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Science 239, 487-491[Medline] [Order article via Infotrieve] |
27. | Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467[Abstract] |
28. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , pp. 9.1-9.62, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
29. | Kudo, S. (1992) Experientia 48, 277-281[Medline] [Order article via Infotrieve] |
30. | Trudel, J., and Asselin, A. (1989) Anal. Biochem. 178, 362-366[Medline] [Order article via Infotrieve] |
31. | Kondo, A., Suzuki, J., Kuraya, N., Hase, S., Kato, I., and Ikenaka, T. (1990) Agric. Biol. Chem. 54, 2169-2170[Medline] [Order article via Infotrieve] |
32. | Shinohara, Y., Sota, H., Kim, F., Shimizu, M., Gotoh, M., Tosu, M., and Hasegawa, Y. (1995) J. Biochem. (Tokyo) 117, 1076-1082[Abstract] |
33. | Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148[Abstract] |
34. |
Hakala, B. E.,
White, C.,
and Recklies, A. D.
(1993)
J. Biol. Chem.
268,
25803-25810 |
35. |
Boot, R. G.,
Renkema, G. H.,
Strijland, A.,
van Zonneveld, A. J.,
and Aerts, J. M.
(1995)
J. Biol. Chem.
270,
26252-26256 |
36. |
Shackelton, L. M.,
Mann, D. M.,
and Millis, A. J.
(1995)
J. Biol. Chem.
270,
13076-13083 |
37. | Kirkpatrick, R. B., Matico, R. E., McNulty, D. E., Strickler, J. E., and Rosenberg, M. (1995) Gene (Amst.) 153, 147-154[CrossRef][Medline] [Order article via Infotrieve] |
38. | Karlsson, R. (1994) Anal. Biochem. 221, 142-151[CrossRef][Medline] [Order article via Infotrieve] |
39. | Adams, D. O., and Hamilton, T. A. (1987) Immunol. Rev. 97, 5-27[Medline] [Order article via Infotrieve] |
40. | Laskin, D. L., and Pendino, K. J. (1995) Annu. Rev. Pharmacol. Toxicol. 35, 655-677[CrossRef][Medline] [Order article via Infotrieve] |
41. | Morrissette, N., Gold, E., and Aderem, A. (1999) Trends Cell Biol. 9, 199-201[CrossRef][Medline] [Order article via Infotrieve] |
42. | Vaday, G. G., and Lider, O. (2000) J. Leukocyte Biol. 67, 149-159[Abstract] |
43. |
Guo, L.,
Johnson, R. S.,
and Schuh, J. C.
(2000)
J. Biol. Chem.
275,
8032-8037 |
44. | Gordon, S. (1999) Immunol. Lett. 65, 5-8[CrossRef][Medline] [Order article via Infotrieve] |
45. | Martinez-Pomares, L., Platt, N., McKnight, A. J., da Silva, R. P., and Gordon, S. (1996) Immunobiology 195, 407-416[Medline] [Order article via Infotrieve] |
46. | Naito, M., Umeda, S., Yamamoto, T., Moriyama, H., Umezu, H., Hasegawa, G., Usuda, H., Shultz, L. D., and Takahashi, K. (1996) J. Leukocyte Biol. 59, 133-138[Abstract] |
47. | Morris, L., Graham, C. F., and Gordon, S. (1991) Development 112, 517-526[Abstract] |
48. |
Owhashi, M.,
Arita, H.,
and Hayai, N.
(2000)
J. Biol. Chem.
275,
1279-1286 |
49. |
Lawson, N. D.,
and Berliner, N.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
10129-10133 |
50. | Owhashi, M., Arita, H., and Niwa, A. (1998) Parasitol. Res. 84, 136-138[CrossRef][Medline] [Order article via Infotrieve] |
51. | Jin, H. M., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Kirkpatrick, R. B., and Rosenberg, M. (1998) Genomics 54, 316-322[CrossRef][Medline] [Order article via Infotrieve] |
52. |
Watanabe, T.,
Suzuki, K.,
Oyanagi, W.,
Ohnishi, K.,
and Tanaka, H.
(1990)
J. Biol. Chem.
265,
15659-15665 |
53. | Henrissat, B., and Bairoch, A. (1993) Biochem. J. 293, 781-788[Medline] [Order article via Infotrieve] |
54. |
Watanabe, T.,
Kobori, K.,
Miyashita, K.,
Fujii, T.,
Sakai, H.,
Uchida, M.,
and Tanaka, H.
(1993)
J. Biol. Chem.
268,
18567-18572 |
55. | Krause, S. W., Rehli, M., Kreutz, M., Schwarzfischer, L., Paulauskis, J. D., and Andreesen, R. (1996) J. Leukocyte Biol. 60, 540-545[Abstract] |
56. | Kirkpatrick, R. B., Emery, J. G., Connor, J. R., Dodds, R., Lysko, P. G., and Rosenberg, M. (1997) Exp. Cell Res. 237, 46-54[CrossRef][Medline] [Order article via Infotrieve] |
57. | Hollak, C. E., van Weely, S., van Oers, M. H., and Aerts, J. M. (1994) J. Clin. Invest. 93, 1288-1292[Medline] [Order article via Infotrieve] |
58. |
Renkema, G. H.,
Boot, R. G.,
Muijsers, A. O.,
Donker-Koopman, W. E.,
and Aerts, J. M.
(1995)
J. Biol. Chem.
270,
2198-2202 |
59. | Millis, A. J., Hoyle, M., Reich, E., and Mann, D. M. (1985) J. Biol. Chem. 260, 3754-3761[Abstract] |
60. |
Boot, R. G.,
Renkema, G. H.,
Verhoek, M.,
Strijland, A.,
Bliek, J.,
de Meulemeester, T. M.,
Mannens, M. M.,
and Aerts, J. M.
(1998)
J. Biol. Chem.
273,
25680-25685 |
61. | Rice, P. A., Goldman, A., and Steitz, T. A. (1990) Proteins 8, 334-340[Medline] [Order article via Infotrieve] |
62. | Fagerstam, L. G., Frostell-Karlsson, A., Karlsson, R., Persson, B., and Ronnberg, I. (1992) J. Chromatogr. 597, 397-410[CrossRef][Medline] [Order article via Infotrieve] |
63. | Karlsson, R., and Stahlberg, R. (1995) Anal. Biochem. 228, 274-280[CrossRef][Medline] [Order article via Infotrieve] |
64. | Shinohara, Y., Hasegawa, Y., Kaku, H., and Shibuya, N. (1997) Glycobiology 7, 1201-1208[Abstract] |
65. |
Lee, Y. C.
(1992)
FASEB J.
6,
3193-3200 |
66. | Crocker, P. R., and Feizi, T. (1996) Curr. Opin. Struct. Biol. 6, 679-691[CrossRef][Medline] [Order article via Infotrieve] |
67. | Stahl, P. D., and Ezekowitz, R. A. (1998) Curr. Opin. Immunol. 10, 50-55[CrossRef][Medline] [Order article via Infotrieve] |
68. |
Shepherd, V. L.,
Freeze, H. H.,
Miller, A. L.,
and Stahl, P. D.
(1984)
J. Biol. Chem.
259,
2257-2261 |
69. |
Ii, M.,
Kurata, H.,
Itoh, N.,
Yamashina, I.,
and Kawasaki, T.
(1990)
J. Biol. Chem.
265,
11295-11298 |
70. |
Koenig, A.,
Norgard-Sumnicht, K.,
Linhardt, R.,
and Varki, A.
(1998)
J. Clin. Invest.
101,
877-889 |
71. | Bernfield, M., Gotte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J., and Zako, M. (1999) Annu. Rev. Biochem. 68, 729-777[CrossRef][Medline] [Order article via Infotrieve] |
72. |
Lindahl, U.,
Kusche-Gullberg, M.,
and Kjellen, L.
(1998)
J. Biol. Chem.
273,
24979-24982 |
73. | Perrimon, N., and Bernfield, M. (2000) Nature 404, 725-728[CrossRef][Medline] [Order article via Infotrieve] |
74. |
Clasper, S.,
Vekemans, S.,
Fiore, M.,
Plebanski, M.,
Wordsworth, P.,
David, G.,
and Jackson, D. G.
(1999)
J. Biol. Chem.
274,
24113-24123 |
75. | Yeaman, C., and Rapraeger, A. C. (1993) J. Cell Biol. 122, 941-950[Abstract] |
76. | Kaltner, H., and Stierstorfer, B. (1998) Acta Anat. (Basel) 161, 162-179[CrossRef][Medline] [Order article via Infotrieve] |
77. | Bevilacqua, M. P. (1993) Annu. Rev. Immunol. 11, 767-804[CrossRef][Medline] [Order article via Infotrieve] |
78. |
Norgard-Sumnicht, K.,
and Varki, A.
(1995)
J. Biol. Chem.
270,
12012-12024 |
79. |
Sun, Y. J.,
Chang, N. C.,
Hung, S. I.,
Chang, A. C.,
Chou, C. C.,
and Hsiao, C. D.
(2001)
J. Biol. Chem.
276,
17507-17514 |