(Received for publication, August 21, 1995; and in revised form, September 11, 1995)
From the
Initial rolling of circulating neutrophils on a blood vessel
wall prior to adhesion and transmigration to damaged tissue is
dependent upon P-selectin expressed on endothelial cells and its
specific neutrophil receptor, the P-selectin glycoprotein ligand-1
(PSGL-1). Pretreatment of neutrophils, HL60 cells, or a recombinant
fucosylated soluble form of PSGL-1 (sPSGL-1.T7) with the cobra venom
metalloproteinase, mocarhagin, completely abolished binding to purified
P-selectin in a time-dependent and EDTA- and diisopropyl
fluorophosphate-inhibitable manner consistent with mocarhagin
selectively cleaving PSGL-1. A polyclonal antibody against the
N-terminal peptide Gln-1-Glu-15 of mature PSGL-1
immunoprecipitated sPSGL-1.T7 but not sPSGL-1.T7 treated with
mocarhagin, indicating that the mocarhagin cleavage site was near the N
terminus. A single mocarhagin cleavage site between Tyr-10 and Asp-11
of mature PSGL-1 was determined by N-terminal sequencing of mocarhagin
fragments of sPSGL-1.T7 and is within a highly negatively charged amino
acid sequence 1-QATEYEYLDYDFLPETEPPE, containing three tyrosine
residues that are consensus sulfation sites. Consistent with a
functional role of this region of PSGL-1 in binding P-selectin, an
affinity-purified polyclonal antibody against residues
Gln-1-Glu-15 of PSGL-1 strongly inhibited P-selectin binding to
neutrophils, whereas an antibody against residues Asp-9-Arg-23
was noninhibitory. These combined data strongly suggest that the
N-terminal anionic/sulfated tyrosine motif of PSGL-1 as well as
downstream sialylated carbohydrate is essential for binding of
P-selectin by neutrophils.
In response to inflammatory stimuli, neutrophils in the adjacent
vasculature initially roll on the blood vessel wall, then stick, and
finally transmigrate to the site of insult(1) . The initial
rolling event involves a class of adhesion proteins termed selectins
(P-, E-, and L-selectin), which mediate the interaction between
leukocytes and endothelial cells by recognizing specific carbohydrate
counterstructures, including sialyl-Lewis
x(2, 3, 4) . P-selectin binds to
10,000-20,000 copies of a single class of binding site on
neutrophils and HL60 cells(4, 5) . Studies in a number
of laboratories have identified a 220-240-kDa, disulfide-linked
homodimeric protein, which appears to specifically bind
P-selectin(6, 7) . This protein is probably identical
to P-selectin glycoprotein ligand-1 (PSGL-1)(
)(8) .
PSGL-1 is a 220-kDa, disulfide-linked homodimeric sialomucin, which,
when expressed in COS cells with the appropriate fucosyltransferase,
binds P-selectin in a similar calcium-dependent manner to the receptor
on neutrophils. PSGL-1 has a signal peptide sequence of 17 amino acids
followed by a 24-amino acid PACE propeptide sequence(8) . The
mature N terminus of PSGL-1 contains an unusual stretch of 20 amino
acids, which is rich in negatively charged aspartate and glutamate
residues and which contains three tyrosine residues that meet the
consensus sequence for O-sulfation by Golgi
sulfotransferase(s)(9) . At least one of these tyrosine
residues is sulfated as evaluated by site-directed mutagenesis and
sulfate labeling experiments. (
)
PSGL-1 also binds
E-selectin. In contrast to P-selectin, however, the requirements for
E-selectin recognition are much less rigid. E-selectin binds a number
of sialomucin and glycoprotein structures if they co-express the
sialyl-Lewis x structure(8, 10) . L-selectin binds to
a number of different counter-receptors, glycoprotein cell adhesion
molecule-1, mucosal addressin cell adhesion molecule-1, and CD34,
which, like PSGL-1, are also sialomucins(11) . A major question
currently unresolved is what determines selectin specificity in the
recognition of specific counter-receptor structures. P-, E-, and
L-selectin are 60-70% homologous in their N-terminal, lectin
motifs, and each similarly recognizes the sialyl-Lewis x and
sialyl-Lewis a carbohydrate structures(11, 12) .
Further, binding of P-selectin to its receptor on neutrophils is
4-5 orders of magnitude more avid than the binding to
sialyl-Lewis x(4, 5, 11, 13) . While
differences in specificity and avidity may in part be accounted for
either by the presentation of multiple sialyl-Lewis carbohydrate
structures on the receptor mucin core or by subtle differences in
carbohydrate structure, it is clear that the protein component of the
sialomucin also plays a critical role in selectin
interaction(8, 14) .
In the present paper, we describe a highly specific metalloproteinase, mocarhagin, which has been purified from the venom of the Mozambiquan spitting cobra, Naja mocambique mocambique. Mocarhagin cleaves a 10-amino acid peptide from the mature N terminus of PSGL-1 and abolishes the ability of PSGL-1 to bind P-selectin. The results are in accord with the negative charge/sulfated tyrosine cluster at the N terminus of PSGL-1 being an important determinant of P-selectin recognition in addition to its recognition of carbohydrate structure.
We have recently identified and purified a novel
metalloproteinase, mocarhagin, from the venom of the Mozambiquan
spitting cobra, N. mocambique mocambique. The proteinase
requires either calcium ion or zinc ion for activity and is fully
inhibited by excess EDTA and by high concentrations of DFP.
Pretreatment of platelets with mocarhagin abolishes their ability to
bind the adhesive ligand, von Willebrand Factor. This is due to
proteolysis between Glu-282 and Asp-283, DEGDTDLYDYYPEEDTEGD, in
the
-chain of the platelet GP Ib-V-IX complex, which occurs as the
sole detectable cleavage on the intact platelet surface.
In the course of these studies, we observed that mocarhagin
was also a potent inhibitor of P-selectin binding to its myeloid
receptor on neutrophils. Pretreatment of either neutrophils or HL60
cells with mocarhagin profoundly and reproducibly affected the
subsequent binding of P-selectin to these treated cells with an
apparent IC of
0.1 µg/ml. A representative
inhibition curve from multiple studies is shown in Fig. 1.
Equivalent data were obtained regardless of whether the
mocarhagin-treated cells were washed or not washed prior to the
addition of P-selectin. Further, inhibition was not reversed by
incubation of the treated cells with fresh medium for up to 3 h.
Finally, mocarhagin had no apparent effect on the molecular size of
P-selectin or on its inherent ability to bind to myeloid cells (data
not shown and see Fig. 2). Treatment of mocarhagin with DFP
completely blocked its ability to inhibit P-selectin binding even at
100 µg/ml (Fig. 1), a result in accord with proteolysis of
the P-selectin receptor. Consistent with this view, the ability of
mocarhagin to inhibit subsequent P-selectin binding was divalent
cation- and time-dependent. If cells were incubated with 12 µg/ml
mocarhagin for 10 s prior to the addition of EDTA and the cells then
washed, P-selectin binding was reduced, even with this brief treatment,
to 40% of normal (data not shown). Cell surface labeling studies,
however, failed to identify a major substrate for mocarhagin on either
neutrophils or HL60 cells (data not shown), a finding consistent with
the exquisite substrate specificity of mocarhagin suggested by the
platelet studies. In addition, the concentrated supernatant from
mocarhagin-treated cells, after removal of mocarhagin by absorption
with heparin-Sepharose CL-6B, did not inhibit binding of P-selectin to
HL60 cells, indicating that a functional fragment of the P-selectin
receptor was not released by mocarhagin treatment.
Figure 1: Effect of mocarhagin on P-selectin binding to neutrophils. Neutrophils were pretreated for 30 min at room temperature with increasing concentrations of mocarhagin (circles) or with mocarhagin that had been treated with DFP (triangles).
Figure 2:
Mocarhagin digestion of soluble P-selectin
glycoprotein ligand. COS-conditioned medium containing
[S]methionine-labeled sPSGL-1.T7 was untreated (lanes 1, 3, 5, 6, and 8)
or digested with 5 µg/ml mocarhagin (lanes 2, 4, 7, and 9). The samples were either directly
electrophoresed (lanes 1 and 2) or precipitated with
the P-selectin IgG chimera LEC
1 (lanes 3 and 4)
or precipitated with LEC
1, which was pretreated with mocarhagin (LEC
1 + mo; lane 5), or immunoprecipitated with
Rb3026 (lanes 6 and 7) or with Rb3443 (lanes 8 and 9).
PSGL-1 has
recently been identified as a functional ligand for P-selectin on HL60
cells(8) . A soluble form of PSGL-1 (sPSGL-1.T7) expressed in
COS cells with an -1,3/1,4-fucosyltransferase also mediates
P-selectin binding in a calcium-dependent manner (8) . One of
the striking features of PSGL-1 is its similarity to the
-chain of
platelet GP Ib. Both are sialomucins and each has immediately
N-terminal to the mucin core a sequence rich in negatively charged
amino acids with three potential sulfated tyrosine
residues(8, 20, 21, 22) . Since
mocarhagin cleaves the
-chain of GP Ib within this negative
charge/sulfated tyrosine cluster (see above), we speculated that
mocarhagin may abrogate P-selectin binding to neutrophils and HL60
cells by cleaving near the N terminus of PSGL-1, a result that would
explain the failure to identify a major substrate for mocarhagin on
myeloid cells. That this is indeed the case is confirmed by the data in Fig. 2. Mocarhagin digestion of PACE-cleaved, fucosylated
sPSGL-1.T7 resulted in only a minor shift, if any, in electrophoretic
mobility of the protein on a SDS-polyacrylamide gel (lanes 1 and 2) but completely abolished the binding of sPSGL-1.T7
to the P-selectin IgG chimera, LEC
1(8) , coupled to
protein A-Sepharose (lanes 3 and 4). To exclude the
possibility that the protease treatment interfered with LEC
1
binding by destroying the LEC
1 protein A-Sepharose complex,
LEC
1-protein A-Sepharose beads were incubated with mocarhagin and
then washed repeatedly to remove any residual protease. The
protease-treated beads were unaffected in their ability to bind
sPSGL-1.T7 (lane 5). Fig. 2also shows the reactivity
of untreated and mocarhagin-digested sPSGL-1.T7 with two polyclonal
antibodies. Rb3026(17) , which was raised against COS-produced
sPSGL-1.T7, precipitated sPSGL-1 independent of mocarhagin digestion (lanes 6 and 7), whereas Rb3443, which was raised
against the N-terminal peptide of PACE-cleaved PSGL-1
(QATEYEYLDYDFLPE), only precipitated untreated sPSGL-1.T7 (lanes 8 and 9), indicating that the N-terminal epitope for Rb3443
is lost after mocarhagin digestion.
N-terminal microsequencing of
purified, mocarhagin-treated sPSGL-1.T7 protein gave the sequence,
DFLPETEPPEML. Mocarhagin thus removes the first 10 amino acids from
PACE-cleaved sPSGL-1.T7. The site of cleavage for mocarhagin between
Tyr-10 and Asp-11 was confirmed using the synthetic peptide,
TEYEYLDYDFLPETE, corresponding to residues 3-17 of mature PSGL-1.
The mocarhagin cleavage sites on PSGL-1 and the -chain of GP Ib
are similar. Each occurs on the N-terminal site of an aspartate residue
and to the C-terminal side of three potential sulfated tyrosine
residues (8, 22) and within an overall negative charge
cluster. Since the proteolytic activity of mocarhagin is inhibited by
heparin,
this preference for negative charge cluster may in
part explain the remarkable substrate specificity of mocarhagin.
Confirmation of the critical importance of the N-terminal sequence of PSGL-1 in P-selectin binding was obtained using anti-peptide antibodies. P-selectin binding to neutrophils was inhibited by 80-90% by an affinity-purified polyclonal antibody against residues Gln-1 to Glu-15 of mature PSGL-1 (QATEYEYLDYDFLPE) but not by an affinity-purified polyclonal antibody against residues Asp-9 to Arg-23 (DYDFLPETEPPEMLR) (Fig. 3) or by non-immune rabbit IgG (not shown). Inhibition by the anti-peptide antibody against Gln-1 to Glu-15 was completely blocked by the presence of either the Gln-1 to Glu-15 or Asp-9 to Arg-23 peptides (data not shown), indicating that anti-peptide antibody, at least in part, recognizes the sequence, Asp-9 to Glu-15.
Figure 3:
Effect of anti-PSGL-1 IgG on P-selectin
binding to neutrophils. Dose-response curves for inhibition of specific
binding of I-P-selectin to neutrophils by polyclonal IgG
against synthetic peptide sequences Gln-1 to Glu-15 (circles)
and Asp-9 to Arg-23 (squares). Data are representative of at
least three experiments with different donor
neutrophils.
Although P-, E-, and L-selectins all recognize similar sialylated carbohydrate structures such as sialyl-Lewis x (11) and many glycoproteins on the surface of myeloid cells contain sialyl-Lewis x (23, 24) , P-selectin appears to be highly specific in its recognition of PSGL-1. The present data suggest that one cause for this specificity is the negative charge/sulfated tyrosine cluster at the N terminus of mature PSGL-1. Proteolytic removal of a N-terminal 10-amino acid peptide by mocarhagin abolished P-selectin binding to PSGL-1 even though this sequence (QATEYEYLDY) is not glycosylated. One explanation for this phenomenon is that removal of this sequence alters the conformational integrity of PSGL-1 such that P-selectin can no longer interact with critical carbohydrate structures associated with the PSGL-1 mucin core. This is unlikely for two reasons. First, an affinity-purified polyclonal antibody against the N-terminal 15 amino acids of mature PSGL-1 also strongly inhibited P-selectin binding to neutrophils. Second, E-selectin also binds to PSGL-1(8, 25, 26) , but, unlike P-selectin, E-selectin binds equally well to mocarhagin-cleaved PSGL-1 (data not shown), suggesting that the carbohydrate recognition structures on PSGL-1 are still inherently accessible. An alternative explanation of the present observations is that P-selectin binding to PSGL-1 is bimodal with P-selectin binding not only involving carbohydrate recognition but also the negative charge/sulfated tyrosine cluster. The approximately 4 order of magnitude difference in avidity for P-selectin binding to sialyl-Lewis x versus receptor (11) is strongly suggestive that additional structural determinants are involved in binding of P-selectin to its myeloid receptor. This is supported by the observation that P-selectin binding to myeloid cells not only depends on the N-terminal lectin domain but also involves the adjacent epidermal growth factor-like motif(27) . We, and subsequently others, have demonstrated that P-selectin binds to heparin and to a wide variety of other sulfated glycans and polyanionic structures(5, 28) . It is tempting to speculate that the N-terminal negative charge/sulfated tyrosine cluster of PSGL-1 represents an equivalent polyanionic recognition site and that the juxtaposition of this sequence with appropriate sialylated carbohydrate structure explains the specificity of P-selectin recognition.