Removal of apoptotic cells is essential for maintenance of tissue homeostasis, organogenesis, remodeling, development, and maintenance of the immune system, protection against neoplasia, and resolution of inflammation. The mechanisms of this removal involve recognition of the apoptotic cell surface and initiation of phagocytic uptake into a variety of cell types. Here we provide evidence that C1q and mannose binding lectin (MBL), a member of the collectin family of proteins, bind to apoptotic cells and stimulate ingestion of these by ligation on the phagocyte surface of the multifunctional protein, calreticulin (also known as the cC1qR), which in turn is bound to the endocytic receptor protein CD91, also known as the α-2-macroglobulin receptor. Use of these proteins provides another example of apoptotic cell clearance mediated by pattern recognition molecules of the innate immune system. Ingestion of the apoptotic cells through calreticulin/CD91 stimulation is further shown to involve the process of macropinocytosis, implicated as a primitive and relatively nonselective uptake mechanism for C1q- and MBL-enhanced engulfment of whole, intact apoptotic cells, as well as cell debris and foreign organisms to which these molecules may bind.

SummaryThis work describes the functional characterization, cDNA cloning, and expression of a novel cell surface protein. This protein designated gClq-R, was first isolated from Raji cells and was found to bind to the globular "heads" of Clq molecules, at physiological ionic strength, and also to inhibit complement-mediated lysis of sheep erythrocytes by human serum. The NHzterminal amino acid sequence of the first 24 residues of the Clq-binding protein was determined and this information allowed the synthesis of two degenerate polymerase chain reaction primers for use in the preparation of a probe in the screening of a B cell cDNA library The cDNA isolated, using this probe, was found to encode a pre-pro protein of 282 residues. The NH2 terminus of the protein isolated from Raji cells started at residue 74 of the predicted pre-pro sequence. The cDNA sequence shows that the purified protein has three potential N-glycosylation residues and is a highly charged, acidic molecule. Hence, its binding to Clq may be primarily but not exclusively due to ionic interactions. The "mature" protein, corresponding to amino acid residues 74-282 of the predicted pre-pro sequence, was overexpressed in Escherichia coli and was purified to homogeneity This recombinant protein was also able to inhibit the complementmediated lysis of sheep erythrocytes by human serum and was shown to be a tetramer by gd filtration in nondissociating conditions. Northern blot and RT-PCR studies showed that the Clq-binding protein is expressed at high levels in Raji and Dandi cell lines, at moderate levels in U937, Molt-4, and HepG2 cell lines, and at a very low level in the HL60 call line. However, it is not expressed in the K562 cell line. Comparison of gClq-R NHz-terminal sequence with that of the receptor for the collagen-like domain ofClq (cClq-R) showed no similarity. Furthermore, antibodies to gClq-R or an 18-amino acid residue-long NHz-terminal synthetic gClq-R peptide did not cross-react with antibodies to cClq-R. Anti-gClq-R immunoblotted a 33-kD Raft cell membrane protein, whereas anti cClq-R recognized a molecule of ,v60 kD. The NH2-terminal sequence of gClg-R appears to be displayed extracdlularly since anti-gClg-R peptide reacted with surface molecules on lymphocytes, polymorphonudear leukocytes, and platelets, as assessed by flow cytometric and confocal laser scanning microscopic analyses. In addition, all or part of the gClq binding domain may reside within the 24 amino acid stretch of the NHz-terminal sequence of gClq-R since the 18 amino acid residue long-synthetic peptide corresponding to this region inhibited serum Clq hemolytic activity The data presented in this report suggest that there are at least two types of Clq-R which appear to be expressed on the same type of ceils and these receptors individually or in concert may contribute to the diversity of Clq-mediated responses.

Efficient apoptotic cell clearance is critical for maintenance of tissue homeostasis, and to control the immune responses mediated by phagocytes. Little is known about the molecules that contribute “eat me” signals on the apoptotic cell surface. C1q, the recognition unit of the C1 complex of complement, also senses altered structures from self and is a major actor of immune tolerance. HeLa cells were rendered apoptotic by UV-B treatment and a variety of cellular and molecular approaches were used to investigate the nature of the target(s) recognized by C1q. Using surface plasmon resonance, C1q binding was shown to occur at early stages of apoptosis and to involve recognition of a cell membrane component. C1q binding and phosphatidylserine (PS) exposure, as measured by annexin V labeling, proceeded concomitantly, and annexin V inhibited C1q binding in a dose-dependent manner. As shown by cosedimentation, surface plasmon resonance, and x-ray crystallographic analyses, C1q recognized PS specifically and avidly (KD = 3.7–7 × 10−8 M), through multiple interactions between its globular domain and the phosphoserine group of PS. Confocal microscopy revealed that the majority of the C1q molecules were distributed in membrane patches where they colocalized with PS. In summary, PS is one of the C1q ligands on apoptotic cells, and C1q-PS interaction takes place at early stages of apoptosis, in newly organized membrane patches. Given its versatile recognition properties, these data suggest that C1q has the unique ability to sense different markers which collectively would provide strong eat me signals, thereby allowing efficient apoptotic cell removal.

InlB is a Listeria monocytogenes protein that promotes entry of the bacterium into mammalian cells by stimulating tyrosine phosphorylation of the adaptor proteins Gab1, Cbl and Shc, and activation of phosphatidyl- inositol (PI) 3-kinase. Using affinity chromatography and enzyme-linked immunosorbent assay, we demonstrate a direct interaction between InlB and the mammalian protein gC1q-R, the receptor of the globular part of the complement component C1q. Soluble C1q or anti-gC1q-R antibodies impair InlB-mediated entry. Transient transfection of GPC16 cells, which are non-permissive to InlB-mediated entry, with a plasmid-expressing human gC1q-R promotes entry of InlB-coated beads. Furthermore, several experiments indicate that membrane recruitment and activation of PI 3-kinase involve an InlB-gC1q-R interaction and that gC1q-R associates with Gab1 upon stimulation of Vero cells with InlB. Thus, gC1q-R constitutes a cellular receptor involved in InlB-mediated activation of PI 3-kinase and tyrosine phosphorylation of the adaptor protein Gab1. After E-cadherin, the receptor for internalin, gC1q-R is the second identified mammalian receptor promoting entry of L. monocytogenes into mammalian cells.

High molecular weight kininogen (HK) and factor XII are known to bind to human umbilical vein endothelial cells (HUVEC) in a zinc-dependent and saturable manner indicating that HUVEC express specific binding site(s) for those proteins. However, identification and immunochemical characterization of the putative receptor site (s) has not been previously accomplished. In this report, we have identified a cell surface glycoprotein that is a likely candidate for the HK (1,2). It possess six domains, the first three homologous to cysteine protease inhibitors (2 and 3 retain inhibitory activity) a fourth domain including the bradykinin moiety, and domains 5 and 6, which interact with surfaces (domain 5) and with prekallikrein or factor XII (domain 6). HK also functions as a coagulation cofactor (3, 4) by virtue of the interactions mediated by domains 5 and 6 (5-7). In addition, HK has been shown to bind to vascular endothelial cells in a zinc-dependent reaction (8, 9) requiring interaction with kininogen domains 3 and 5 (10, 11). The isolated heavy and light chains derived from kinin-free kininogen can each interact with endothelial cells (12) because they contain domains 3 and 5, respectively, although the two sites may act cooperatively in the intact protein (13).We have also shown that the initiating protein of the cascade, factor XII or Hageman factor, can also bind to endothelial cells with a Km (144 nM) and zinc requirement (50 ,tM) virtually identical to that of HK (14). Furthermore, HK and factor XII compete for binding (Ki of 98-152 nM), which suggests interaction with a common receptor (14).In the present report, we describe the isolation and partial characterization of the putative high molecular weight kininogen receptor from human umbilical vein endothelial cells (HUVEC). This molecule is a 33-kDa membrane protein that binds to HK or factor XII in a zinc-dependent reaction. Immunochemical and partial sequence analyses show this protein to be identical to the receptor that binds to the globular "heads" of Clq (gClq-R) described by Ghebrehiwet et al. (15). MATERIALS AND METHODSEndothelial Cell Culture. HUVECs were isolated according to Jaffe (16) and cultured in gelatin-coated dishes in Medium 199 (GIBCO) with 20% fetal bovine serum (FBS), 20% NU-serum (Collaborative Research), 100 ,tg/ml penicillin, 100 ,ug/ml streptomycin, and 2.5 ,ug/ml amphotericin B. For cell-binding studies, the cells were subcultured into gelatincoated 96-well strip plates in 0.2 ml culture medium and the cells were washed 3x in phosphate-buffered saline (PBS) to remove bound serum proteins before each experiment. The cells were identified as endothelial cells by their distinct cobblestone morphology and interaction with antiserum to Von Willebrand's factor (16). All cells were used at the third cell passage.Preparation of the HK-Affinity Column. Purified HK (Enzyme Research Laboratories, South Bend, IN) was coupled with 3M Emphaze Biosupport Medium AB1 (Pierce) according to the manufacturer's directions. Briefly, 10 mg ...

Platelets participate in a variety of responses of the blood to injury. An emerging body of evidence suggests that these cells express an intrinsic capacity to interact with and trigger both classical and alternative pathways of complement. This activity requires cell activation with biochemical agonists and/or shear stress, and is associated with the expression of P-selectin, gC1qR, and chondroitin sulfate. Platelet mediated complement activation measurably increases soluble inflammatory mediators (C3a and C5a). Platelets may also serve as targets of classical complement activation in autoimmune conditions such as antiphospholipid syndromes (APS) and immune thrombocytopenia purpura (ITP). Retrospective correlation with clinical data suggests that enhanced platelet associated complement activation correlates with increased arterial thrombotic events in patients with lupus erythematosus and APS, and evidence of enhanced platelet clearance from the circulation in patients with ITP. Taken together, these data support a role for platelet mediated complement activation in vascular inflammation and thrombosis.

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Human gC1q-R (p33, p32, C1qBP, TAP) is a ubiquitously expressed, multiligand-binding, multicompartmental cellular protein involved in various ligand-mediated cellular responses. Although expressed on the surface of cells, an intriguing feature of the membrane-associated form of gC1q-R is that its translated amino acid sequence does not predict the presence of either a sequence motif compatible with a transmembrane segment or a consensus site for a glycosylphosphatidylinositol anchor. Moreover, the N-terminal sequence of the pre-pro-protein gC1q-R contains a motif that targets the molecule to the mitochondria and as such was deemed unlikely to be expressed on the surface. However, several lines of experimental evidence clearly show that gC1q-R is present in all compartments of the cell, including the extracellular cell surface. First, surface labeling of B lymphocytes with the membrane-impermeable reagent sulfosuccinimidyl 6-(biotinamido)hexanoate shows specific biotin incorporation into the surface-expressed but not the intracellular form of gC1q-R. Second, FACS and confocal laser scanning microscopic analyses using anti-gC1q-R IgG mAb 60.11 or 74.5.2, and the fluorophore Alexa 488-conjugated F(ab')2 goat anti-mouse IgG as a probe, demonstrated specific staining of Raji cells (>95% viable). Three-dimensional analyses of the same cells by confocal microscopy showed staining distribution that was consistent with surface expression. Third, endothelial gC1q-R, which is associated with the urokinase plasminogen activator receptor, and cytokeratin 1 bind 125I-high molecular weight kininogen in a specific manner, and the binding is inhibited dose-dependently by mAb 74.5.2 recognizing gC1q-R residues 204-218. Fourth, native gC1q-R purified from Raji cell membranes but not intracellular gC1q-R is glycosylated, as evidenced by a positive periodic acid Schiff stain as well as sensitivity to digestion with endoglycosidase H and F. Finally, cross-linking experiments using C1q as a ligand indicate that both cC1q-R and gC1q-R are co-immunoprecipitated with anti-C1q. Taken together, the evidence accumulated to date supports the concept that in addition to its intracellular localization, gC1q-R is expressed on the cell surface and can serve as a binding site for plasma and microbial proteins, but also challenges the existing paradigm that mitochondrial proteins never leave their designated compartment. It is therefore proposed that gC1q-R belongs to a growing list of a class of proteins initially targeted to the mitochondria but then exported to different compartments of the cell through specific mechanisms which have yet to be identified. The designation 'multifunctional and multicompartmental cellular proteins' is proposed for this class of proteins.