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.
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