(Received for publication, August 2, 1995; and in revised form, October 5, 1995)
From the
C1q, the recognition subunit of the classical complement pathway, interacts with specific cell surface molecules via its collagen-like region (C1q-CLR). This binding of C1q to neutrophils triggers the generation of toxic oxygen species. To identify the site on C1q that interacts with the neutrophil C1q receptor, C1q was isolated, digested with pepsin to produce C1q-CLR, and further cleaved with either trypsin or endoproteinase Lys-C. The resulting fragments were separated by gel filtration chromatography and analyzed functionally (activation of the respiratory burst in neutrophils) and structurally. Cleavage of C1q-CLR with endoproteinase Lys-C did not alter its ability to trigger neutrophil superoxide production. However, when C1q-CLR was incubated with trypsin under conditions permitting optimal cleavage, the ability of C1q-CLR to stimulate superoxide production in neutrophils was completely abrogated. Fractionation of the digests obtained with the two enzymes and identification by amino acid sequencing permitted localization of the receptor interaction site to a specific region of the C1q-CLR. Circular dichroism analyses demonstrated that cleavage by trypsin does not denature the remaining uncleaved collagen-like structure, suggesting that after trypsin treatment, the loss of activity was not due to a loss of secondary structure of the molecule. However, irreversible heat denaturation of C1q-CLR also abrogated all activity. Thus, a specific conformation conferred by the collagen triple helix constitutes the functional receptor interaction site. These data should direct the design of future specific therapeutic reagents to selectively modulate this response.
C1q, the recognition subunit of the classical complement
pathway, is a 460,000-Da serum protein that has an unusual
macromolecular structure and contributes to a variety of functions in
the response of the host to infection or injury. As part of the C1
complex, C1q binds to immune complexes or antibody-independent C1
activators resulting in the initiation of the classical complement
cascade(1) . However, upon dissociation from the
C1rC1s
tetramer(2) , C1q can interact
with cell surface molecules via its collagen-like domain, inducing
cell-specific responses. One example of such a C1q receptor-mediated
response is the enhancement of phagocytic capacity (3, 4, 5) that occurs when monocytes and
culture-derived macrophages interact with C1q. Furthermore, interaction
of C1q with neutrophils (6) , eosinophils(7) , and
vascular smooth muscle cells (8) triggers the generation of
bactericidal oxygen species.
C1q shares an unusual macromolecular
structure with certain other molecules also known to enhance uptake of
specific pathogenic material (9, 10, 11, 12, 13) . Like
C1q, both pulmonary surfactant protein A (SP-A) ()and
mannose-binding protein (MBP) have collagen-like sequences contiguous
with noncollagen-like sequences, short NH
-terminal domains
containing interchain disulfide bonds, and a single disruption in the
Gly-X-Y repeat pattern within the collagen-like sequence (13, 14, 15) causing a characteristic
``kink'' or bend in the tertiary structure. Like C1q, SP-A (9) and MBP (16) can enhance Fc receptor- and
complement receptor-mediated phagocytosis. However, neither SP-A nor
MBP by themselves stimulate superoxide
(O
) production by polymorphonuclear
leukocytes (17) in contrast to the readily detectable
stimulation by C1q(6, 17) . This suggests that the
ligand interaction sites of the C1q receptor that triggers superoxide
production (C1qR
Very little is currently
known about the ligand requirements for functional interaction with the
C1q receptor on any cell types. It is known that the 176,000-Da
collagen-like fragment of Clq mediates the functional interaction with
most cells(4, 20) , that C1rC1s
blocks that interaction(6) , and that heat-aggregated C1q
loses the ability to trigger B cell immunoglobulin
secretion(21) . In addition, it appears that a multivalent
interaction is required for C1q to trigger a response upon binding to
the cells(6, 21) . It is not known whether this is due
to a requirement for receptor clustering or a surface- or
aggregate-induced conformational alteration. This requirement is
similar to that seen with CR1, CR2, Fc
RIII, and many
other receptors(22, 23) . If the receptor for C1q that
triggers superoxide production differs from the one that enhances
phagocytosis, it is probable that the interaction sites on the ligand,
C1q, that mediate these responses are also distinct. Therefore,
identification of the receptor interaction sites should permit the
selective manipulation of the responses mediated by the different
receptors; that is, to enhance phagocytosis without generating
extracellular superoxide or to inhibit the production of superoxide
without effecting monocyte phagocytic capacity.
The present
investigation characterizes a specific region on the collagen-like
domain of C1q (C1q-CLR) that interacts with the neutrophil C1q receptor
(C1qR
Preliminary experiments demonstrated that optimal
conditions for trypsin digestion of C1q-CLR were achieved when
TPCK-trypsin was added to C1q-CLR (2.5-4.5 mg/ml in TBS, pH 7.5)
to produce an enzyme:substrate ratio (E:S) of 1:10 (w:w) and incubated
at 50 °C for 30 min. Endo Lys-C was incubated with C1q-CLR at
similar concentrations in TBS, pH 7.5 (E:S, 1:200, w:w) at 37 °C
for 2 h. Digestions were terminated by placing the samples on ice.
Aliquots of the samples were then electrophoresed under nonreducing
conditions using the Tricine SDS-PAGE system (10% acrylamide) described
by Schägger and von Jagow(29) . Fragments
produced by enzymatic digestion were characterized and separated by gel
filtration chromatography using a Superose 12 HR 10/30 (Pharmacia
Biotech Inc.) column equilibrated in TBS, pH 7.4 (in the presence or
the absence of 5 mM CaCl). The void volume of the
column was 8.1 ml, and the total column volume was 25 ml. Circular
dichroism was recorded at ambient temperature using a Jasco J720
spectropolarimeter. Data were collected at 0.5-nm intervals, and 4
scans were averaged but not smoothed. A cell of 0.5-mm path length was
used. Some circular dichroism spectra were obtained using a Jasco 500C
circular dichrometer with a temperature-controlled cuvette. Protein
concentrations used were 0.4-1 mg/ml. All data were converted to
mean residue ellipticity.
Figure 1:
Effect of heat treatment on the
circular dichroism spectra of C1q-CLR. A, mean residue
ellipticity at 224 nm of C1q-CLR in 10 mM acetic acid
(), TBS (
), or TBS plus 5 mM Ca
(
) was recorded as a function of temperature. B,
complete CD spectra of C1q-CLR heated at the noted temperatures for 30
min followed by cooling for greater than 1 h to allow refolding. MRE is the mean residue ellipticity in degrees (deg)
cm
dmol
.
To determine to what extent, if any, this denaturation was reversible and if this denaturation altered the ability of C1q-CLR to trigger neutrophil superoxide production, C1q-CLR was incubated at pH 7.2 for 30 min at 56, 79, and 100 °C, temperatures known to induce increasingly greater loss of specific secondary structure, and then cooled to ambient temperature. The CD of the cooled material was then compared with that of an unheated control sample to determine if renaturation had occurred. In contrast to that seen with the acidic conditions used by Brodsky-Doyle and colleagues (35) (and repeated by us), the CD spectra of C1q-CLR heated to 56 °C at pH 7.2 and subsequently recooled, was essentially identical to that of the untreated control, with the exception of some variability in the negative peak at 197 nm (Fig. 1B). This renaturation after heating was complete after an incubation of 45-60 min on ice. Heating the protein to 79 °C, however, resulted in a greater and largely irreversible loss of C1q-CLR secondary structure. Samples heated at 79 °C and above did not renature even after days at 4 °C (data not shown).
The effect of loss of secondary structure
on the ability of C1q-CLR to trigger the generation of superoxide by
neutrophils was then examined. Whereas irreversible loss of secondary
structure by heating to 79 or 100 °C resulted in complete loss of
the ability of C1q-CLR to trigger neutrophil
O production, C1q-CLR heated to 56
°C, followed by the recovery of secondary structure upon cooling,
retained total functional activity as compared with the untreated
C1q-CLR in its capacity to trigger O
(Fig. 2). In this experiment and all superoxide assays
performed, neutrophils in uncoated control wells produced no
superoxide, with a recording essentially identical to the 79 °C/100
°C samples (data not shown).
Figure 2:
Effect of loss of native secondary
structure due to heat treatment on the ability of C1q-CLR to
functionally interact with the neutrophil
C1qR. C1q-CLR was either
left untreated (solid line, 4 °C) or heated to 56 °C (dashed line), 79 °C (dotted line), or 100 °C (dashed and dotted line) and allowed to cool for at least 45
min. The protein concentration was then adjusted to 30 µg/ml and
tested for the ability to trigger superoxide production by neutrophils
as described under ``Materials and
Methods.''
Figure 3: Trypsin cleaves all three chains of C1q-CLR. SDS-PAGE analysis under nonreducing conditions of C1q-CLR (lanes 1 and 5), mock digested C1q-CLR (lanes 2 and 6), and after trypsin digestion (lanes 3 and 7) before (lanes 1-3) and after (lanes 5-7) purification via fast performance liquid chromatography gel filtration. Samples in lanes 6 and 7 are from the peak fractions designated by the arrows in Fig. 4(A and B, respectively). Relative molecular mass markers are in lane 4.
Figure 4: Gel filtration demonstrates that C1q-CLR is digested with trypsin. Typical Superose 12 column profile of C1q-CLR (1 mg) incubated at 50 °C for 30 min without enzyme (A) or with trypsin (B) at E:S of 1:10. C is the profile of trypsin alone at the same concentration used in the digestion. The main fragment (designated by the open arrows) was used in the structural and functional assays. The areas labeled a, b, and c in B indicate the fractions pooled for amino acid sequencing and referred to in Table 1. (The second peak in A is a nonprotein contaminant variably detected in fast protein liquid chromatography profiles.) The flow rate of the column was 0.5 ml/min.
The major peak of both the undigested
C1q-CLR and the trypsin-generated NH-terminal fragment of
C1q-CLR (noted with the arrows in Fig. 4) were then
assayed for the ability to trigger polymorphonuclear leukocyte
superoxide production. The fragment resulting from trypsin digestion
had very little activity as compared with the untreated or mock
digested control (Fig. 5). This loss of activity did not reflect
an inability of the trypsin-generated fragment to bind to the
microtiter well surface as nearly equal molar amounts of the
trypsin-digested, and untreated C1q-CLR were detected both by protein
assay and by a more sensitive enzyme-linked immunosorbent assay using
an anti-C1q monoclonal antibody. The data were consistent with loss of
a critical sequence(s) required for the interaction with
C1qR
Figure 5:
Trypsin digestion abrogates the
stimulation of O generation by C1q-CLR.
Neutrophils were added to microtiter wells coated with 100 µg/ml of
the protein in the main peak from the Superose purification of C1q-CLR
that was untreated (solid line), mock digested (dashed and
dotted line), or digested with trypsin (E:S, 1/10) (dotted
line). Superoxide production by neutrophils was assayed by
measuring reduction of cytochrome c. The data presented are
from one experiment representative of
three.
Amino acid sequencing demonstrated that the earliest eluting peak
from the trypsin-treated sample (Fig. 4B, pool
a) contained the intact NH termini of the A and C
chains. The lack of the B chain NH
terminus is consistent
with the previous identification of pyroglutamate at the NH
terminus of this chain(36) . Treatment with
pyroglutamate-aminopeptidase prior to NH
-terminal sequence
analysis allowed the quantitative identification of the predicted B
chain residues Leu-Ser-(Cys)-Thr and thus verified that the B chain
NH
terminus was present but blocked by this modified
glutamine. Thus, the loss of activity was not due to digestion of the
amino terminus of any of the three chains of the C1q molecule.
Furthermore, no evidence of any cleavage products associated with the
main (NH
-terminal) fragment after purification was
detected, nor were trypsin autodigestion fragments contaminating this
main protein peak. No cleavages by trypsin occurred in the first 31
amino acids of the A or C chain as sequencing through the first
NH
-terminal 31 residues on the A and C chains demonstrated
a quantitative yield of each amino acid following the arginine and
lysine residues (Fig. 6). Amino acid sequencing of the other
fractions separated by the Superose column containing peptides of
smaller size (Fig. 4B, pools b and c)
allowed identification of multiple trypsin cleavage sites in the
C1q-CLR and provided evidence that the most NH
-terminal
cleavage occurred on the A chain after Arg
, on the B chain
after Lys
, and on the C chain after Arg
( Table 1and Fig. 6). When tested for the ability to trigger
superoxide production using the standard assay conditions, none of the
fractions containing the smaller peptide fragments demonstrated any
superoxide generating activity (data not shown).
Figure 6:
Schematic model and amino acid sequences
of fragments derived as a result of enzymatic digestion of C1q-CLR. A, localization of the most NH-terminal trypsin (black arrows) and endo Lys-C (black arrowheads)
cleavage sites on the A, B, and C chains of C1q. The open
arrowheads indicate the kink region of C1q. Breaks in the A, B,
and C chain indicated at amino acids 97, 96, and 92 designate the most
NH
-terminal cleavage by pepsin (which yields the intact
C1q-CLR). B, amino acid sequences of fragments derived by
enzymatic digestion. The amino acid sequences detected by
NH
-terminal amino acid sequencing of the intact C1q-CLR- (small vertical dashes), trypsin- (thick short
dashes), or endo Lys-C- (solid thick line) digested
C1q-CLR and a cyanogen bromide-generated fragment of C1q (thick
long dashes). Data from both total digest and from purified pools
after separation of total digests by Superose chromatography are
included. The sequence is taken from Sellar et
al.(30) . Amino acids are indicated using the single
letter code, with hydroxyproline and hydroxylysine (as determined by
Sellar et al.(30) ) being indicated by the lowercase p and k, respectively. According to Reid
and Thompson(36) , all hydroxylysines are glycosylated
(glucose-galactose disaccharide) except at positions B 50 and B 65. The
NH
terminus of the B chain of C1q-CLR was derived after
treatment with
pyroglutamate-aminopeptidase.
Analysis by SDS-PAGE
demonstrated that specific peptide bonds of the C1q-CLR molecules were
quantitatively and homogeneously cleaved by endo Lys-C at an E:S of
1:200 (Fig. 7). No additional cleavage occurred when higher
amounts (E:S, 1:20) of endo Lys-C were added (data not shown) or when
C1q-CLR was preheated to 50 °C prior to digestion (Fig. 7, lane 4). The major proteolytic components were then separated
and purified by Superose size exclusion chromatography.
NH-terminal sequencing of the first eluting peak (Fig. 8B, pool a, corresponding to Fig. 7, lane 1) demonstrated that as in the trypsin
digest, this peak contained an unaltered NH
terminus with
no cleavage fragments remaining associated with the major C1q-CLR
fragment generated. Sequencing of the later eluting peak areas (Fig. 8B, pools b and c) as well as
the total digest identified all of the endo Lys-C fragments generated ( Table 1and Fig. 6). The most NH
-terminal
cleavage sites that identify the COOH terminus of the major fragment
generated by endo Lys-C and purified by gel chromatography were at
Lys
of the A chain, Lys
of the B chain, and
Lys
of the C chain. The NH
-terminal C1q-CLR
fragment generated by endo Lys-C retained complete activity in the
neutrophil superoxide assay (Fig. 9), in contrast to the
observed loss of activity of the fragment generated by trypsin
digestion (Fig. 5).
Figure 7: Endo Lys-C cleaves all three chains of C1q-CLR. SDS-PAGE analysis of mock digested C1q-CLR (lanes 2 and 6) and C1q-CLR after digestion with endo Lys-C (lanes 1, 4, and 5) and before (lanes 4, 5, and 6) and after (lanes 1 and 2) purification via Superose gel filtration. Samples in lanes 1 and 2 are from the peak fractions designated by the arrows in Fig. 8(B and A, respectively). The sample in lane 4 was preheated at 50 °C for 30 min prior to the standard incubation for 2 h at 37 °C. For comparison, trypsin-digested C1q-CLR is shown in lane 7. Molecular mass markers are in lane 3. All samples were electrophoresed under nonreducing conditions.
Figure 8: Gel filtration of C1q-CLR after endo Lys-C digestion. C1q-CLR incubated at 37 °C for 2 h without enzyme (A) or with endo Lys-C at an E:S of 1:200 (B) was applied to Superose 12 column and eluted as described. The main fragment (designated by the arrows), eluting at 10.7 ml, was used in all structural and functional assays. The areas labeled a, b, and c in B indicate the fractions pooled for amino acid sequencing and referred to in Table 1. Flow rate of the column was 0.5 ml/min.
Figure 9:
Endo
Lys-C digestion has no inhibitory effect on the stimulation of
O generation by C1q-CLR. Superoxide
production by neutrophils was assayed by measuring reduction of
cytochrome c. The fragment obtained from C1q-CLR digested with
endo Lys-C (E:S, 1:200) (dotted line) retains full activity
relative to the untreated C1q-CLR (solid line) or mock
digested C1q-CLR (dashed and dotted line). Protein
concentration used to coat the wells was 30
µg/ml.
Figure 10:
Enzymatic cleavage of C1q-CLR by trypsin
or endo Lys-C does not alter the collagen-like secondary structure of
the resulting fragments. Circular dichroism spectra of the main
fragment obtained from tryptic digestion (A) or from endo
Lys-C digestion (B) of C1q-CLR show the typical profile of a
collagen-like structure (maximum, 223 and minimum,
200 nm).
Compared with the control without enzyme (
), the digested
fragment (
) maintains the original collagen-like structural
characteristics even after the significant loss of cleaved peptides. MRE, mean residue ellipticity in degrees (deg)
cm
dmol
.
Structural and functional studies presented here demonstrate
that the region of the C1q collagen-like domain that interacts with a
neutrophil surface receptor to initiate superoxide production is
present on the fragment generated by endo Lys-C digestion of C1q-CLR
but is lost upon digestion with trypsin. Amino acid sequencing of the
fragments generated by each of these enzymes localized the most
NH-terminal cleavage sites for endo Lys-C to the COOH
terminus of Lys
of the A chain, Lys
of the B
chain, and Lys
in the C chain, whereas trypsin cleaved on
the COOH-terminal of Arg
of the A chain, Lys
of the B chain, and Arg
of the C chain. CD analyses
of the fragments after protease digestion demonstrated that peptide
cleavage under these conditions, even close to the kink region, does
not alter, to a major extent, the remainder of the collagen-like
secondary structure. Thus, it appears that the
C1qR
Initial studies of heat-denatured C1q-CLR demonstrated
that the ability of C1q to interact with the neutrophil C1q receptor
and trigger superoxide production required some degree of collagen
helical secondary structure ( Fig. 1and Fig. 2). Brodsky
and colleagues previously presented data in which different synthetic
collagen-like peptides were analyzed by both CD and equilibrium
sedimentation(37) . Comparison of our CD data with their more
extensive characterization of peptide structure would suggest that the
decrease in the magnitude of both the positive (223 nm) and the
negative peaks (197 nm) seen after heating C1q-CLR to 70 °C or
higher is due to a loss of triple helical content within the C1q-CLR
subunits. There is no evidence for a significant population of single
chain molecules, as there is no discernible shift in the position of
the peaks detected by CD. These results suggest that the
C1qR
Figure 11: Molecular model of residues 38-61 of the A, B, and C chain of the C1q collagen-like domain required for superoxide production. The model of C1q (model B in from Kilchherr et al.(39) ) was used to place the C1q A, B, and C chains in register. The C1q A, B, and C chain residues were superimposed on coordinates of the three-dimensional structure of a collagen triple helix peptide consisting of proline-hydroxyproline-glycine repeats (38) using the Biopolymer module in the Biosym Insight molecular modeling package. The amino acid side chains were rotated to match the well established favored rotamers for side chains.
The region of the
C1q molecule shown in this report to be of functional importance for
triggering superoxide production in the neutrophil is different from
that proposed by Malhotra et al.(43) to be the site
of interaction of C1q with the calreticulin-like 56,000 M C1q-binding protein, designated as cC1qR (C1q
receptor interacting with the collagen-like region of C1q). These
investigators deduced that the region on the NH
-terminal
side of the kink in the C1q-CLR was the interaction site for cC1qR
based on the comparisons of amino acid sequences and charge
distributions among members of the family of molecules referred to as
``defense collagens'' (44) or
``collectins''(45) . This family of molecules,
including MBP, SP-A, conglutinin, and C1q, have collagen-like sequence
domains and usually display lectin-like activity(46) . However,
MBP and SP-A have not yet been shown to independently trigger
O
production as does C1q, and there has
been no evidence reported that cC1qR is the receptor through which
immobilized C1q triggers superoxide production. Therefore, it is likely
that C1qR
MBP and SP-A, like C1q, have been shown
to stimulate the ingestion of pathogens (47, 48, 49) and enhance the phagocytic
capacity of monocytes and macrophages(4, 9) .
Monoclonal antibodies to a 126,000 M cell surface
molecule (distinct from the calreticulin-like cC1qR) have been isolated
that inhibit the MBP-(16) , SPA-, (
)and C1q-mediated
enhancement of phagocytosis(18, 19) . These antibodies
do not inhibit the production of superoxide by
neutrophils(19) , providing further evidence that the C1q
receptor that modulates phagocytic activity on myeloid cells and
C1qR
In 1983, Schumaker and co-workers (51) postulated a model of
C1 macromolecular structure in which the tetramer
C1rC1s
associates with the central portion of
the C1q molecule within the cone defined by the C1q arms with the ends
of C1r
C1s
wrapping around the arms of the C1q
hexamer at the region just above the kink region. This model was based
on electron microscopy, molecular volume calculations, and symmetry
considerations. The data presented here that localizes the neutrophil
receptor interaction site above the kink support this model of
Schumaker and co-workers (51) because our previous data
demonstrated that the addition of C1r and C1s to C1q to form the
complete C1 complex blocks the ability of C1q to stimulate superoxide
production(6) . These data then are consistent with the close
association of C1r
C1s
with the region between
residues 40-60 rather than with the region closer to the globular
heads.
Finally, NH-terminal amino acid sequencing of the
intact C1q molecule and fragments produced by enzymatic digestion ( Table 1and Fig. 6) allowed the confirmation of several
previously reported sequences throughout the collagen-like region of
C1q (31) . (
)The sites of pepsin cleavage on each
chain were clearly defined as in these preparations the COOH-terminal
sequences were obtained in high yield without evidence of partial
cleavages. In addition, we established the presence of a hydroxylated
proline at position 75 in the A chain. Proline had been predicted by
the cDNA sequence (30) but differed from the reported residue
determined by amino acid sequencing(31) . In addition, the
lysines at amino acid 10 of chain A and at residue 19 of chain C
reported by Reid (31) were obtained in a lower yield than
expected and thus can be assumed to be partially hydroxylated.
Interestingly, the peptide bond after the hydroxylysine at position B
65 was only partially cleaved by trypsin but was quantitatively cleaved
by endo Lys-C. Although this was the only hydroxylysine that was
cleaved by these proteases, its susceptibility was not due to
heterogeneity of the post-translational hydroxylation, because there
was no evidence of a lysine detected upon amino acid sequencing.
Whereas, unlike most other hydroxylysines in C1q-CLR, this residue is
unglycosylated(31) , carbohydrate-dependent steric hindrance
cannot be the only factor determining cleavage, because the
hydroxylysine at position B 50 is also not glycosylated but was never
seen to be cleaved by either trypsin or endo Lys-C.
In summary, C1q
functions both as the recognition molecule of C1, the initiator of the
classical complement pathway, and as an element in the first line of
defense (52, 53) by triggering the activation of the
NADPH oxidase in neutrophils(17) , resulting in the production
of microbicidal oxygen radicals, and by enhancing the efficiency of
phagocytic cells to clear pathogenic material. Because, as discussed
above, the C1q receptor that modulates phagocytic activity on myeloid
cells and C1qR