(Received for publication, August 31, 1994; and in revised form, December 6, 1994)
From the
Heparan sulfate proteoglycans at cell surfaces or in extracellular matrices bind diverse molecules, including growth factors and cytokines, and it is believed that the activities of these molecules may be regulated by the metabolism of heparan sulfate. In this study, purification of a heparan sulfate-degrading enzyme from human platelets led to the discovery that the enzymatic activity resides in at least two members of the platelet basic protein (PBP) family known as connective tissue activating peptide-III (CTAP-III) and neutrophil activating peptide-2. PBP and its N-truncated derivatives, CTAP-III and neutrophil activating peptide-2, are CXC chemokines, a group of molecules involved in inflammation and wound healing. SDS-polyacrylamide gel electrophoresis analysis of the purified heparanase resulted in a single broad band at 8-10 kDa, the known molecular weight of PBP and its truncated derivatives. Gel filtration chromatography of heparanase resulted in peaks of activity corresponding to monomers, dimers, and tetramers; these higher order aggregates are known to form among the chemokines. N-terminal sequence analysis of the same preparation indicated that only PBP and truncated derivatives were present, and commercial CTAP-III from three suppliers had heparanase activity. Antisera produced in animals immunized with a C-terminal synthetic peptide of PBP inhibited heparanase activity by 95%, compared with activity of the purified enzyme in the presence of the preimmune sera. The synthetic peptide also inhibited heparanase by 95% at 250 µM, compared to the 33% inhibition of heparanase activity by two other peptides. The enzyme was determined to be an endoglucosaminidase, and it degraded both heparin and heparan sulfate with optimal activity at pH 5.8. Chromatofocusing of the purified heparanase resulted in two protein peaks: an inactive peak at pI 7.3, and an active peak at pI 4.8-5.1. Sequence analysis showed that the two peaks contained identical protein, suggesting that a post-translational modification activates the enzyme.
The dynamic role of heparan sulfate proteoglycans (HSPGs) ()in biology has become increasingly apparent(1) .
HSPGs are proteins with linear chains of repeating disaccharides,
called glycosaminoglycans, and are found in abundance in the
extracellular matrix and on cell surfaces. HSPGs participate in diverse
processes, including blood coagulation, the anchoring of enzymes in the
vascular lumen, and in cell adhesion and growth. Endothelial
cell-associated HSPGs bind and potentiate antithrombin III,
contributing to a nonthrombogenic vascular surface(2) .
Vascular HSPGs also bind many enzymes, including lipoprotein lipase,
elastase, and superoxide dismutase, anchoring and modulating the
function of these enzymes in the vascular
lumen(3, 4, 5) . HSPGs modulate cell-matrix
adhesion by interacting with matrix proteins, such as vitronectin,
fibronectin, laminin, and collagen(6, 7) . HSPGs also
modulate cell-cell adhesion. HSPGs serve as ligands for L-selectin and
P-selectin, adhesion molecules expressed on leukocytes, and platelets
that are responsible for the initial loose attachment to endothelial
cells(8) . Additionally, HSPGs bind and sequester cytokines and
can present the bound cytokines to the loosely adhered
leukocytes(9) . These cytokines can trigger events required for
the tight adhesion and extravasation of the leukocytes. The HSPGs in
the extracellular matrix/basement membrane provide a readily available
tissue storage depot for growth factors and cytokines(10) .
Finally, heparanase activity releases growth factors from their heparan
sulfate storage sites in the extracellular matrix(11) ,
providing a mechanism for the induction of growth and migration of
diverse cell types in normal or pathologic situations(11) .
Since HSPGs are now recognized as active biologic modulators, their degradation would be expected to have significant regulatory consequences. Indeed, HSPG catabolism is observed in inflammation, wound repair, diabetes, and cancer metastasis, suggesting that enzymes that degrade heparan sulfate play important roles in pathologic processes. Heparanase activity has been described in activated immune system cells and cancer cells, but research has been handicapped by the lack of biologic tools to explore potential causative roles of heparanase in disease. Although bacterial heparanases have been purified, earlier attempts to isolate mammalian heparanases have yielded only partial purifications and/or a lack of specific information such as amino acid sequences. In this paper, we report the purification of a mammalian heparanase from activated human platelet supernatants and demonstrate that the enzymatic activity resides in neutrophil activating peptide-2 (NAP-2) and connective tissue activating peptide-III (CTAP-III), two processed forms of the CXC chemokine platelet basic protein (PBP).
Preparation of heparan
[S]sulfate glycosaminoglycan from
[
S]HSPG was achieved by
-elimination.
[
S]HSPG (250,000 cpm) was incubated overnight
with 1 M NaBH
, 50 mM NaOH at 45 °C.
The glycosaminoglycan was isolated by chromatography on Superose 6 HR
10/30 in PBS.
Chondroitin [S]sulfate
glycosaminoglycan was prepared from human embryonic lung fibroblasts.
Cells were cultured overnight with 0.1 mCi/ml sodium
[
S]sulfate, and the conditioned medium was
collected and lyophilized. The lyophilizate was suspended in 6 M urea, 0.15 M NaCl, 20 mM Tris-HCl, pH 7.0, and
chromatographed on a Mono Q HR5/5 column using a 40-ml linear gradient
between 0.15 and 1.15 M NaCl in 6 M urea, 20 mM Tris-HCl, pH 7.0. Aliquots of each fraction were assayed for
radioactivity, and fractions containing the radiolabel that eluted from
the column between 0.85 and 1.15 M NaCl were pooled; 1
lactalbumin hydrolysate carrier was added, and the chondroitin
[
S]sulfate was precipitated with 100% EtOH as
described above. The precipitate was dissolved in 4 M guanidine hydrochloride, 20 mM Tris-HCl, pH 7.2, and
chromatographed on a Superose 12 HR 10/30 column equilibrated in the
same buffer. Aliquots of each fraction were used to determine
radioactivity, and the fractions containing radioactivity that eluted
with a calculated molecular mass >70 kDa were pooled and dialyzed
against PBS.
For determination of the optimal pH for heparanase activity, the 0.1 M sodium acetate buffer was replaced by 50 mM citrate, citrate-phosphate, or phosphate buffer at varying pH values. For samples from chromatographic steps performed under reducing conditions (1 mM GSH, 1 mM DTT), the concentration of a thiol oxidant (diamide) needed for optimum activity was determined. This concentration (100 µM diamide) was added to all assay tubes when reduced samples were assayed.
Verification that the antisera specifically recognized commercial
CTAP-III (2.5 µg, Celsus Laboratories), isolated heparanase (1.5
µg), and 7-10-kDa proteins in 10 µl of the platelet
supernatant used for the heparanase isolation was achieved by Western
blotting. The proteins or platelet supernatant were separated on a
reducing 18% polyacrylamide gel and transferred to nitrocellulose or
polyvinylidene difluoride using a semi-dry transfer unit (Hoeffer
Semi-Phor TE70). Western blotting was performed at 25° C according
to the following protocol: 1 h Blocking Buffer (5% milk, 0.05%
Tween-20, 0.15 M NaCl, 20 mM Tris-HCl, pH 7.0), 2 h
preimmune or antisera (1:1500 in Blocking Buffer), 3 15-min
rinses in Blocking Buffer, 1 h peroxidase labeled goat anti-chicken IgG
(1:500 in Blocking Buffer), 3
15-min rinses in PBS, and 30 s
membrane development with the TMB membrane peroxidase substrate system.
For antisera neutralization experiments, the preimmune and antisera
were exchanged into PBS using a 100-kDa cut-off membrane in order to
remove low molecular weight chicken heparanase normally present in the
serum. Aliquots of isolated heparanase (15 ng) were preincubated for 30
min with 2 µl of either preimmune or antisera before adding the
[S]HSPG to determine heparanase activity.
Figure 3:
N-terminal sequence of purified heparanase
and CXC chemokines. An aliquot of heparanase from each of two Step 5
preparations was desalted using a M 5000 membrane
and was subjected to N-terminal sequencing as described under
``Experimental Procedures.'' The published sequence for
platelet basic protein is reviewed by Miller and Krangel(31) ,
and N-terminal cleavages producing PBP, CTAP-III,
-thromboglobulin, and NAP-2 are indicated by arrows.
Calculated masses of the derivatives are based on 1 amino acid =
110.5 Da. The asterisk denotes a blank sequencing
cycle.
Figure 9: A view of a smoothed backbone model of a NAP-2 dimer. The backbone of the model is identical to that of the parent IL-8 structure from which it was constructed, as described under ``Experimental Procedures.'' Positive and negative amino acid residues are shown in blue and red, respectively. The side chains of possible catalytic residues (Glu-93, Asp-107, and Asp-109) are highlighted in yellow.
Figure 1:
Heparin-Sepharose and cation exchange
chromatography of heparanase from activated platelet supernatants. A, heparin-Sepharose chromatography. Heparanase was eluted
under reducing conditions (1 mM GSH, 1 mM DTT) with a
linear NaCl gradient () at pH 5 as described under
``Experimental Procedures.'' 8-ml fractions were collected,
and a 10-µl aliquot was assayed for heparanase activity (
).
Activity that eluted between 0.9 M and 1.15 M NaCl
(indicated by solidbar on lowerpanel) was used for further purification. B,
cation-exchange chromatography. The flow-through from the DEAE-Sephacel
column was adjusted to pH 5, loaded onto a column of Poros HS/F, and
eluted with a linear NaCl gradient (
), as described under
``Experimental Procedures.'' 3-ml fractions were collected,
and 10-µl aliquots were assayed for heparanase activity
(
).
The final yield of heparanase protein from 1850 ml of platelet-rich plasma was 2.7 mg. The overall recovery of activity was 8%, with a 4150-fold purification (Table 1). The preparation was judged to be homogeneous by silver-staining of SDS-PAGE (Fig. 2, inset).
Figure 2:
Molecular size and oligomerization of
human platelet heparanase. An aliquot of human platelet heparanase was
chromatographed on Superdex 75 under reducing conditions and assayed
for heparanase activity as described under ``Experimental
Procedures.'' Elution positions of standards are indicated at the top of the figure: bovine serum albumin (66 kDa), carbonic
anhydrase (29 kDa), trypsin inhibitor (20.1 kDa), -lactalbumin
(14.2 kDa), and aprotinin (6.5 kDa). Inset, an aliquot of Step
5 heparanase was desalted using M
5000 membranes
and electrophoresed under reducing conditions on an 18%
SDS-polyacrylamide gel. Migration positions of molecular mass markers
are shown on the left of the inset: bovine serum
albumin (69 kDa), ovalbumin (45 kDa), soybean trypsin inhibitor (21
kDa), myoglobin I + II (14.4 kDa), myoglobin I (8.1 kDa), and
myoglobin II (6.2 kDa).
Figure 4:
Degradation of S-HSPG by
commercial CTAP-III. Increasing amounts of commercial CTAP-III were
assayed for heparanase activity. Shown are the results of a 16-h assay
with protein obtained from Calbiochem. Similar results were obtained
using protein obtained from either Celsus Laboratories or Haematologic
Technologies.
To rule out the possibility that the
heparanase activity was attributable to a minor contaminant and not to
CTAP-III, we prepared antisera from synthetic peptides of CTAP-III, and
tested the ability of these antisera to neutralize heparanase activity.
Keyhole limpet hemocyanin conjugates of C-terminal
(CNQVEVIATLKDGRKICLDPDAPRIKKIVQKK) and N-terminal (NLAKGKEE-SLDSDLC)
synthetic peptides of CTAP-III/NAP-2 (residues 89-120 and
44-57 in Fig. 3, respectively) were used for immunizing
chickens. These antisera, but not the preimmune sera, bound
specifically to 8-10-kDa proteins on immunoblots of SDS-PAGE
separations of purified platelet heparanase and commercial CTAP-III
(data not shown). An experiment was designed to measure the heparanase
activity of the purified enzyme in the presence of preimmune or
antisera. In the presence of the preimmune sera, the enzyme had 14.3
± 0.1 units of heparanase activity, while in the presence of the
C-terminal peptide antisera, only 0.8 ± 0.2 units of heparanase
were detected (p < 0.001; results confirmed in a second
experiment). The N-terminal peptide antiserum was not able to
neutralize the heparanase activity. Similar results were obtained when
the synthetic peptides themselves were tested for their ability to
neutralize heparanase activity. Heparanase assays conducted with 3
nM enzyme, 47 nM [S]HSPG
substrate, and varying concentrations of peptides showed that
heparanase activity was inhibited by 95% in the presence of 250
µM C-terminal peptide. By contrast, heparanase activity in
the presence of 250 µM of either the N-terminal peptide or
an unrelated peptide (PLALWAR) was inhibited by only 33%. The ability
of both the C-terminal peptide or antisera from a chicken immunized
with the C-terminal synthetic peptide to neutralize heparanase activity
demonstrates conclusively that CTAP-III and NAP-2 possess heparanase
activity, and suggests that the C-terminal region may be essential for
catalysis.
Figure 5:
Chromatofocusing of platelet heparanase.
An aliquot of Step 5 heparanase was chromatofocused on PBE 94 between
pH 7.3 and 4.0, as described under ``Experimental
Procedures.'' Fractions of 2 ml were collected, and the pH
() and heparanase activity (
) for each fraction was
plotted. Data shown are respresentative of three
experiments.
Figure 6:
Enzymatic degradation of intact
[S]HSPG and heparan
[
S]sulfate chains by platelet heparanase.
Sepharose 6B chromatography was used to monitor the lower molecular
weight products formed during an incubation of 28,000 cpm of intact
>70 kDa [
S]HSPG (A) or heparan
[
S]sulfate chains (B) for 16 h
with (
) or without (
) the Step 5 heparanase, or for 45 min
with HNO
(
), as described under ``Experimental
Procedures.'' One-ml fractions were collected, and the
radioactivity was quantitated by liquid scintillation counting. The arrow denotes the included volume of the column, determined
with [
S]sulfate. C, lower molecular
weight products formed during an incubation of >70 kDa
[
S]HSPG with the Step 5 heparanase for 3 h
(
), 6 h (
), or 21 h (
) were detected by centrifugation
through 30-kDa membranes, described under ``Experimental
Procedures.'' Each point represents the mean ± S.D. for
triplicates. Graphs shown are representative of at least four
determinations.
The buffer
used for determination of heparanase activity contains six different
protease inhibitors, so it was unlikely that the S-labeled
fragments produced by heparanase digestion resulted from cleavage of
the protein backbone of the heparan sulfate proteoglycan. To provide
further evidence that this hypothesis was correct,
S-heparan sulfate glycosaminoglycan (no protein backbone)
was prepared from the [
S]HSPG by
-elimination and was used as the substrate for a heparanase
digestion. Fig. 6B shows that the Step 5 enzyme was
able to cleave the protein-free
S-labeled heparan sulfate
glycosaminoglycan to intermediate-sized fragments, confirming that the
enzyme is a glycosidase.
A second method of detecting the presence
of S-labeled low molecular weight fragments produced by
digestion of heparanase with the >70 kDa
[
S]HSPG was to centrifuge the digest through
30-kDa membranes. The percent of radioactivity that passed through the
30-kDa membrane was used as a measure of heparanase activity. Fig. 6C shows an example of this type of assay using
increasing amounts of the Step 5 enzyme for 3, 6, or 21 h. Increased
heparanase activity corresponded with increased protein concentration
and time. Since analysis of heparanase activity using this method was
rapid, reproducible, and allowed simultaneous analysis of several
digests, this method was routinely used to assay for heparanase
activity throughout the isolation of heparanase.
The platelet
heparanase was determined to be an endoglucosaminidase. To show this,
heparin and heparan sulfate, prereduced with sodium borohydride at
alkaline pH, were incubated for 24 h with Step 5 enzyme under
conditions designed to achieve complete enzymatic cleavage. After 24 h,
the sample was lyophilized, the new reducing ends produced by enzymatic
cleavage were labeled with sodium [H]borohydride,
and the unincorporated sodium [
H]borohydride was
separated from the saccharides by gel filtration on Bio-Gel P2 (Fig. 7A). Cleavage of both heparin and heparan sulfate
by the platelet heparanase resulted principally in disaccharides. The
disaccharides were lyophilized and subjected to total acid
hydrolysis/nitrous acid deamination to produce monosaccharides, which
were separated on phosphocellulose paper chromatography under
conditions that distinguish the derivatives of sugars found in heparin
and heparan sulfate (uronic acid, glucuronic acid, and glucosamine). As
shown in Fig. 7B, the
[
H]monosaccharide derived from heparan sulfate
that had been cleaved with heparanase and labeled with sodium
[
H]borohydride eluted in a position similar to
the glucosamine standard, thus identifying this platelet heparanase as
an endoglucosaminidase. Similar results were obtained when the heparin
was the glycosaminoglycan under study (data not shown). The Step 5
enzyme was unable to cleave [
S]chondroitin
sulfate (data not shown).
Figure 7:
Gel filtration and paper chromatography of
heparin and heparan sulfate after cleavage by heparanase and labeling
with sodium [H]borohydride. A, Bio-Gel
P2 chromatography of heparan sulfate (upper) and heparin (lower) after cleavage by heparanase and labeling with sodium
[
H]borohydride, as described under
``Experimental Procedures.'' Two-ml fractions were collected,
and 100 µl was used to determine radioactivity by liquid
scintillation counting. Positions of native heparin and disaccharides
produced by HNO
digestion of heparin were determined by a
carbazole assay, and [
H]mannose was determined by
liquid scintillation counting. B, cellulose phosphate paper
chromatography of labeled monosaccharides obtained from the labeled
disaccharide produced by enzymatic digestion and borotritide labeling.
Chromatography and preparation of standards is described under
``Experimental Procedures.'' The upperpanel shows the results obtained with monosaccharides obtained from
heparan sulfate. The lowerpanel shows the elution
positions of gulonic acid derived from glucuronic acid (
), idonic
acid derived from iduronic acid (
), and anhydromannitol derived
from glucosamine (
). Data shown are representative of two
independent experiments.
The pH optimum of the heparanase was determined by conducting activity assays between pH 3.5 and 8.0, using a citrate buffer (pH 3.5-6.0), citrate-phosphate buffer (pH 6.5-7.0), and phosphate buffer (pH 7.5-8.). Heparanase was active between pH 5.0 and 7.5, with the optimum pH at 5.8 (Fig. 8). The enzyme rapidly and irreversibly lost activity above pH 7.5.
Figure 8:
The HSPG-degrading activity of isolated
platelet heparanase as a function of pH. Heparanase activity was
assayed by incubating the [S]HSPG with 10 µl
of Step 5 heparanase for 16 h at varying pH values. Assays were
performed as described under ``Experimental Procedures,''
except that the 0.1 M sodium acetate, pH 5.6, buffer was
replaced with 0.05 M citrate buffer between pH 3.5 and 6.0;
0.05 M citrate-phosphate buffer between pH 6.5 and 7.0; or
0.05 M phosphate buffer between pH 7.5 and 8.0. Data shown are
representative of three separate
experiments.
This report is important in that it describes the purification and identification of a mammalian heparanase, an enzyme that has been difficult to isolate. Furthermore, this is the first demonstration of enzymatic activity in a member of the CXC chemokine family.
One of the more striking observations from these studies is the small molecular mass of this heparanase. That the mass of heparanase is below 10 kDa may account for our low recovery after heparin-Sepharose chromatography, which included concentration and buffer exchange through 10-kDa membranes. Subsequent isolations utilized membranes with a 2-kDa cut-off. Furthermore, in our earlier unsuccessful attempts to isolate a heparanase, we routinely used 10% SDS-polyacrylamide gels, which would not resolve proteins with molecular masses below 20 kDa. Other estimations of the molecular mass of heparanase in nonhomogeneous preparations, measured by gel filtration chromatography, are from 50 to 134 kDa(23, 24, 25) . CTAP-III/NAP-2 also exist as higher order aggregates, and aggregation may account for the various higher estimates of molecular mass made by other researchers.
Another observation is the amount of heterogeneity displayed by this protein. At least four different N-truncated forms of this protein have been isolated, and the protein exists as monomers, dimers, and tetramers. Moreover, there is heterogeneity in its isoelectric point, which others have also observed(26) . SDS-PAGE results in a broad band from 8.5 to 10.1 kDa, consistent with the molecular mass of processed forms of PBP. Other researchers have found that storage increases the amounts of processed forms(27) . We found 100% NAP-2, a highly processed form, in a second Step 5 preparation that used activated platelets that had been stored frozen for several months. Gel filtration chromatography of the Step 5 heparanase resulted in peaks of enzymatic activity that corresponded to higher order aggregates. Analysis of the Step 5 enzyme by chromatofocusing showed variations in the isoelectric point of the protein, suggesting that a post-translational modification that lowers the pI is responsible for the enzymatic activity. The sequence of PBP contains potential sites for protein kinase C phosphorylation (Ser-35, Ser-36, Thr-41, Ser-70, Thr-87, and Thr-97), N-myristoylation (Gly-86), amidation (Asp-100), and O-linked glycosylation (Ser-70), but no sites for N-linked glycosylation. Of these potential modifications, phosphorylation is attractive, since it would lower the pI and is known to contribute to the regulation of many enzymes. In our experience, gradient elution of this protein from various chromatographic matrices results in either more than one protein peak (heparin-Sepharose) or in broad peaks (Poros S). This may be the result of variations in the isoelectric point or aggregation state. This large reservoir of potential microheterogeneity may have complicated the isolation of active heparanase in the past and may make it difficult to express this protein recombinantly. Native, commercial CTAP-III displayed heparanase activity, but with a specific activity reduced at least 100-fold. Commercial recombinant NAP-2 produced in E. coli did not have activity (data not shown). This lower specific activity or lack of activity may be accounted for by either the methods used to isolate the protein, including chromatography above pH 7.5, reverse phase chromatography with trifluoroactic acid/acetonitrile, and lyophilization, or by expression in systems that would not be expected to correctly fold or modify the protein. We are now in the process of producing the protein in mammalian expression systems.
Because the purification to homogeneity and characterization of mammalian heparanases has been difficult, other purified heparanases are not available for comparison. Most reports contain only partial descriptions. In one of the more well characterized reports, Oosta et al.(23) described the purification of a human platelet heparanase that was homogeneous with respect to charge by acidic disc gel electrophoresis, although determination of homogeneity by SDS-PAGE or N-terminal sequencing was not reported. The protein was estimated to have a molecular mass of 134 kDa by gel filtration chromatography and was found to be an endoglucuronidase, not an endoglucosaminidase as shown for our platelet heparanase. Since amino acid sequence data are not available for the earlier protein, sequence comparison is not possible. A melanoma heparanase, whose molecular size of 94 kDa and 25 N-terminal amino acid residues are identical to glucose-regulated protein/endoplasmin, has also been described to be an endoglucuronidase (24) , although the presence of heparanase activity in this protein has recently been challenged(28) . It is entirely possible that platelets, as well as other cells, contain several heparanases that are regulated differently and/or have different specificities. Three bacterial heparinases from Flavobacterium heparinum have been isolated and characterized(29) . They are similar to the present enzyme in that they also are endoglucosaminidases, but the bacterial enzymes have molecular masses of 42.5, 84, and 70 kDa, and the present heparanase shares no amino acid homology with the one bacterial heparanase of known amino acid sequence(30) .
The most provocative
information from this study is that members of the PBP family are
heparan sulfate/heparin-degrading enzymes. PBP and naturally occurring N-truncated derivatives of PBP are members of the large
chemokine (chemoattractant and cytokine) superfamily (for review, see (31) ) which functions in inflammation, wound healing, and
growth regulation. The superfamily includes platelet factor 4, PBP and N-truncated derivatives, IL-8, GRO-,
,
, 9E3, ENA-78, IP-10, macrophage inflammatory proteins 1
(MIP-1
), MIP-1
, MIP-2, monocyte chemoattractant proteins-1
(MCP-1) MCP-2, MCP-3, and RANTES. All family members are basic
heparin-binding proteins with molecular masses below 12 kDa. The
members share similar tertiary structures and are known to form dimers
and tetramers, consistent with our gel filtration data showing that the
isolated protein exists as higher order aggregates.
Since a high
degree of sequence identity is shared among members of the chemokine
family, it is possible to model the three-dimensional structure of
NAP-2 based on the NMR solution structure of IL-8/NAP-1 (17, 18) . We derived an approximate NAP-2 dimer model
by a pairwise replacement of residues, which differed between NAP-2 and
IL-8/NAP-1 (Fig. 9). Two antiparallel -helices can be seen
corresponding to the C-terminal regions of each monomer. Truncation of
these
-helices in IL-8 led to the loss of heparin-binding
activity(32) , and it is probable that the same heparin-binding
function exists for the C-terminal
-helices in NAP-2. It is
tempting to speculate as to what amino acids might play a role in
catalysis. As a group of enzymes, most glucosidases function via an
acid-base catalytic mechanism analogous to that for lysozyme where a
Glu and an Asp participate in the hydrolysis (for review, see (33) ). Interestingly, 2 Asp and 1 Glu appear at each end of
the groove defined by the pair of
-helices in NAP-2. Accordingly,
some combination of these Asp and Glu residues (residues 93, 107, and
109 in Fig. 4; highlighted in Fig. 9) could serve as
catalytic residues in the breakdown of heparan sulfate. These residues
are contained within the C-terminal peptide that was effective in
neutralizing heparanase activity. This model may be useful in
predicting whether other chemokines may be expected to have heparanase
activity. Among PBP, IL-8, GRO, 9E3, MCPs, MIPs, PF4, RANTES, IP-10,
and LD78, only PBP and its derivatives contain both Glu-93 and Asp-109
and would be expected to be active if these residues are involved in
catalysis. If Glu-93 and Asp-107 conferred activity, then PBP, IL-8,
9E3, MIP-2, and MCP-1 may be expected to have heparanase activity.
Most of the research described for the chemokines has focussed on the mechanisms responsible for their chemoattractant and activating abilities. CTAP-III has also been described as a mitogen for a variety of cell types(34) , but the mechanism by which it promotes growth is not well understood. That the chemokine CTAP-III has the ability to degrade heparan sulfate suggests a novel molecular mechanism that may account for the growth promoting activities of CTAP-III. Heparanase cleavage of immobilized HSPGs in the extracellular matrix could promote growth by allowing the heparan sulfate and any bound growth factors to become soluble and interact with growth factor receptors on adjacent cells.
The dual function of CTAP-III/NAP-2 as
both a heparanase and a neutrophil chemoattractant suggests that a
complex relationship may exist where each activity could alter the
activity or bioavailability of the other function in various
pathologies. For example, chemokines are known to be immobilized by
proteoglycans, which can present the chemokines to inflammatory
cells(9) . The heparanase activity of the chemokine could
down-regulate inflammation by degrading focal sites of chemokine
anchoring on the surface of the inflamed endothelium. In vascular
pathologies, postinjury adherence of platelets to the subendothelium
causes platelet activation and release of granule contents, including
20 µg of PBP (and derivatives) per 10 cells(34) . Degradation of vascular heparan sulfate by
CTAP-III would remove antithrombin III binding sites and promote
thrombogenesis. NAP-2 may attract neutrophils, consistent with the
concept that pathophysiological events associated with arterial injury
are controlled by multiple cell types and pathways, including
neutrophils(35) . In some situations, such as the inflamed
synovial joint, where CTAP-III and NAP-2 are found in
abundance(36, 37) , it is difficult to predict the
consequences of having both activities. For example, others have
reported that chemokine activity is enhanced following binding to
heparin(32) . It could be envisioned that built-in heparanase
activity would degrade the heparin ``enhancer'' and
down-regulate the activity of the chemokine. It could also be
envisioned that chemokines are stored in the extracellular matrix in a
fashion analogous to growth factors (10) and that heparanase
activity would mobilize and therefore make the chemokine more
available. In tumor growth and metastasis, it is also difficult to
predict the effects of the dual functions. Heparanase activity may
degrade the heparan sulfate needed for the binding of growth factors to
high affinity receptors (38) and inhibit growth. Alternatively,
the dual functions may work synergistically to both attract leukocytes
and to mobilize growth factors from extracellular matrix storage sites.
In regard to these apparent paradoxes, an essential question is whether
the heparanase activity is a critical part of the mitogenic and/or
chemoattractant activity ascribed to CTAP-III/NAP-2 or if the
enzymatic, mitogenic, and chemoattractant functions on this single
protein function independently.
HSPGs associate with diverse biologically active molecules. The enzymatic release of bioactive complexes may be a critical regulatory mechanism in controlling the availability of these molecules. Our observations that the previously known proinflammatory chemokines CTAP-III and NAP-2 are heparanases suggest that this class of proteins may have multiple mechanistic capabilities and biologic functions, and this may profoundly influence our understanding of vascular biology.