Identification and partial characterization of human platelet C1q binding sites

We recently showed that human blood platelets bind the complement component, C1q, and mAb directed against lymphoblastoid C1q receptors in a specific and saturable manner. To identify and further characterize platelet C1q binding sites, human platelets were washed, solubilized in Triton X-100 and either subjected to SDS-PAGE and Western blotting by using monoclonal (II1/D1) and polyclonal antibodies recognizing C1qR on lymphoblastoid cells, or applied to a C1q-Sepharose affinity column under low ionic strength conditions (20 mM NaCl). Adherent proteins were eluted with buffer containing 300 mM NaCl. Western blotting with monoclonal and polyclonal antibodies directed against C1qR showed exclusive reactivity with a 67,000 m.w. protein possessing intrachain disulfide bonds. SDS-PAGE of C1q-Sepharose eluates also revealed the presence of a 67,000 protein which was accompanied to varying degrees by a 94,000 constituent. Because similar m.w., 125I-labeled proteins were recovered from C1q-Sepharose columns to which lysed, surface-labeled platelets had been applied, both 94,000 and 67,000 components appear to be platelet membrane constituents. The 94,000 and 67,000 species, however, appear to be antigenically distinct. The 94,000 protein was immunoprecipitated by polyclonal antibodies against platelet membrane glycoprotein IIIa but not polyclonal antibodies against C1qR, whereas the 67,000 protein was immunoprecipitated exclusively by the polyclonal anti-C1qR antibody. The 67,000 protein thus appears to represent platelet C1q binding sites resembling C1qR on lymphoblastoid cells.     

Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and (19)F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ((5F)W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that (5F)W incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when (5F)W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. (19)F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each (5F)W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.     

By-passing selection: direct screening for antibody-antigen interactions using protein arrays

We have developed a system to identify highly specific antibody-antigen interactions by protein array screening. This removes the need for selection using animal immunisation or in vitro techniques such as phage or ribosome display. We screened an array of 27 648 human foetal brain proteins with 12 well-expressed antibody fragments that had not previously been exposed to any antigen. Four highly specific antibody-antigen pairs were identified, including three antibodies that bind proteins of unknown function. The target proteins were expressed at a very low copy number on the array, emphasising the unbiased nature of the screen. The specificity and sensitivity of binding demonstrates that this 'naive' screening approach could be applied to the high throughput isolation of specific antibodies against many different targets in the human proteome.     

New antibody immobilization method via functional liposome layer for specific protein assays

A specific protein assay system based on functional liposome-modified gold electrodes has been demonstrated. To fabricate such assay system, a liposome layer was initially grown on top of a gold layer. The liposome layer contained two kinds of functional molecules: biotin molecules for the binding sites of streptavidin and N-(10,12-pentacosadiynoic)-acetylferrocene molecules for the facile redox probe in electrochemical detections. Then, streptavidin was attached on the functional liposme-modified layer using the interaction of streptavidin-sbiotin complex. On the streptavidin-attached surface, antibody molecules, anti-human serum albumin antibodies could be immobilized without any secondary antibodies. AFM imaging of the streptavidin-attached liposome surface revealed a uniform distribution of closely packed streptavidin molecules. In situ quartz-crystal microbalance and electrochemical measurements demonstrated that the wanted antibody-antigen reactions should occur with high specificity and selectivity. Our specific antibody assay system, based on a functional liposome modified electrode, can be developed further to yield sophisticated structures for numerous protein chips and immunoassay sensors.     

The mechanism of inhibition of the activation of the complement first component by polyanions and polycations

Polyethyleneimine (PEI, 50 kDa) and polymethacrylic acid (PMA, 200 kDa) were shown to inhibit the lysis of sheep erythrocytes induced by the guinea pig complement. They twofold suppress the hemolysis at the concentrations of 0.47 and 0.89 microgram/ml, respectively. The inhibitory effect on the binding of the C1q subunit of human complement to the sensitized sheep erythrocytes (EA) was found to depend on the component of the reaction with which the inhibitors were preliminarily incubated. When an inhibitor, C1q, and EA were simultaneously incubated, the inhibition constants for PEI and PMA were 17 +/- 6 and 8.1 +/- 0.1 micrograms/ml, respectively. The preincubation of EA with PEI and the subsequent washing out of the inhibitor resulted in the inhibition constant of 22 +/- 3 micrograms/ml. No inhibitory effect was observed after a similar preincubation of EA with PMA. No inhibition was also detected when the inhibitors were added after the formation of the C1q complex with antibodies. These observations suggest that the binding of antibodies to cationic PEI prevents the C1q-antibody complex formation, while the binding of anionic PMA to the active site of C1q impedes the interaction of this subunit with immunoglobulins. Moreover, within the range of concentrations studied, the studied inhibitors did not affect the subsequent C1q binding to the C1r and C1s enzymes.     

Phagocytic cell molecules that bind the collagen-like region of C1q. Involvement in the C1q-mediated enhancement of phagocytosis

C1q binds to and elicits cellular responses by several cell types, including monocytes, macrophages, neutrophils, B cells, and fibroblasts. The cell-binding domain is located within the collagen-like pepsin-resistant region of the C1q molecule (C1q tails). An affinity matrix of C1q tails coupled to Sepharose was used to select C1q-binding proteins from detergent extracts of surface-iodinated human monocytes, polymorphonuclear leukocytes, and the U937 cells. The major radiolabeled polypeptide eluted specifically from the ligand affinity column had an apparent molecular mass (Mr) of 126,000. Minor iodinated components eluted from Sepharose-tails migrated with Mr of 216,000 and 55,000. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions no change in the migration of any of these polypeptide bands was detected. None of these polypeptides reacted with antibodies directed against the integrins alpha 5 beta 1 (fibronectin receptor) or alpha v beta 3 (vitronectin receptor), LFA-1, or to several other cell adhesion molecules. The Mr 126,000 band was found to contain more than one polypeptide. Lectin binding properties, susceptibility to glycosidases and proteases, and immunoreactivity with the monoclonal antibody L-10, indicated that CD43 (sialophorin/leukosialin) is a component of this band. However, further data show that a monoclonal antibody, generated by immunization with the isolated Clq-binding fractions, recognizes a cell surface sialoglycoprotein distinct from CD43 and inhibits the C1q-mediated enhancement of phagocytosis in monocytes. These latter observations provide the first definitive connection between a specific phagocytic cell surface protein and a known C1q-mediated function. While these proteins contain sialic acid, binding assays and functional assays using neuraminidase-treated cells demonstrate that the functional interaction between C1q and the cell surface is not via sialic acid. The data taken together indicate either that the functional C1q receptor on phagocytic cells is a multi-subunit complex or that multiple proteins can interact with the fragment of C1q containing the cell-binding domain, at least one of which is involved in the C1q-mediated enhancement of phagocytosis.     

Platelet C1q receptor interactions with collagen- and C1q-coated surfaces

We recently described specific binding sites for C1q on human blood platelets. Structural similarities between the amino-terminal of C1q and collagen have suggested that receptors for both molecules on platelets might be the same. The present study thus compared the interaction of purified C1q receptors (C1qR) and whole platelets with collagen- and C1q-coated polystyrene surfaces. Surfaces coated with BSA or gelatin served as controls. Purified 125I-labeled C1qR recognized both C1q- and collagen-coated surfaces in a divalent, cation-independent manner. This adhesion was inhibited by polyclonal or monoclonal (II1/D1) anti-C1qR antibodies. Although C1qR adhered preferentially to C1q-coated surfaces, adhesion to bovine and human type I collagen, as well as to human type III and V collagen, was also noted. In parallel studies, 51Cr-labeled platelets bound equally well to collagen- or C1q-coated surfaces, albeit in a magnesium-dependent manner. Partial inhibition of platelet adhesion was observed in the presence of RGDS, despite the inability of RGDS to modify C1qR interaction with C1q or collagen. Moreover, anti C1qR antibodies selectively inhibited platelet adhesion to C1q-coated surfaces, whereas antibodies specific for the GPIa/IIa collagen receptor (6F1) preferentially inhibited platelet collagen interactions. These data support the presence of distinct platelet membrane C1qR, which may cross-react with collagen, and suggest that C1qR are necessary but not sufficient for platelet adhesion to C1q-coated surfaces. Additional divalent cation and/or RGD-sensitive binding sites may participate.     

Contribution of chondroitin sulfate A to the binding of complement proteins to activated platelets

Background

Exposure of chondroitin sulfate A (CS-A) on the surface of activated platelets is well established. The aim of the present study was to investigate to what extent CS-A contributes to the binding of the complement recognition molecule C1q and the complement regulators C1 inhibitor (C1INH), C4b-binding protein (C4BP), and factor H to platelets.

Principal Findings

Human blood serum was passed over Sepharose conjugated with CS-A, and CS-A-specific binding proteins were identified by Western blotting and mass spectrometric analysis. C1q was shown to be the main protein that specifically bound to CS-A, but C4BP and factor H were also shown to interact. Binding of C1INH was dependent of the presence of C1q and then not bound to CS-A from C1q-depleted serum. The specific interactions observed of these proteins with CS-A were subsequently confirmed by surface plasmon resonance analysis using purified proteins. Importantly, C1q, C4BP, and factor H were also shown to bind to activated platelets and this interaction was inhibited by a CS-A-specific monoclonal antibody, thereby linking the binding of C1q, C4BP, and factor H to exposure of CS-A on activated platelets. CS-A-bound C1q was also shown to amplify the binding of model immune complexes to both microtiter plate-bound CS-A and to activated platelets.

Conclusions

This study supports the concept that CS-A contributes to the binding of C1q, C4BP, and factor H to platelets, thereby adding CS-A to the previously reported binding sites for these proteins on the platelet surface. CS-A-bound C1q also seems to amplify the binding of immune complexes to activated platelets, suggesting a role for this molecule in immune complex diseases.     

Rationalization and design of the complementarity determining region sequences in an antibody-antigen recognition interface

Protein-protein interactions are critical determinants in biological systems. Engineered proteins binding to specific areas on protein surfaces could lead to therapeutics or diagnostics for treating diseases in humans. But designing epitope-specific protein-protein interactions with computational atomistic interaction free energy remains a difficult challenge. Here we show that, with the antibody-VEGF (vascular endothelial growth factor) interaction as a model system, the experimentally observed amino acid preferences in the antibody-antigen interface can be rationalized with 3-dimensional distributions of interacting atoms derived from the database of protein structures. Machine learning models established on the rationalization can be generalized to design amino acid preferences in antibody-antigen interfaces, for which the experimental validations are tractable with current high throughput synthetic antibody display technologies. Leave-one-out cross validation on the benchmark system yielded the accuracy, precision, recall (sensitivity) and specificity of the overall binary predictions to be 0.69, 0.45, 0.63, and 0.71 respectively, and the overall Matthews correlation coefficient of the 20 amino acid types in the 24 interface CDR positions was 0.312. The structure-based computational antibody design methodology was further tested with other antibodies binding to VEGF. The results indicate that the methodology could provide alternatives to the current antibody technologies based on animal immune systems in engineering therapeutic and diagnostic antibodies against predetermined antigen epitopes.     

Analysis of protein interaction and function with a 3-dimensional MALDI-MS protein array

Protein profiling and characterization of protein interactions in biological samples ultimately require indicator-free methods of signal detection, which likewise offer an opportunity to distinguish specific interactions from nonspecific protein binding. Here we describe a new 3-dimensional protein microchip for detecting biomolecular interactions with matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS); the microchip comprises a high-density array of methacrylate polymer elements containing immobilized proteins as capture molecules and directly interfaces with a commercially available mass spectrometer. We demonstrated the performance of the chip in three types of experiments by detecting antibody-antigen interactions, enzymatic activity, and enzyme-inhibitor interactions. MALDI-MS biochip-based tumor necrosisfactor alpha (TNF-alpha) immunoassays demonstrated the feasibility of detecting antigens in complex biological samples by identifying molecular masses of bound proteins even at high nonspecific protein binding. By detecting model interactions of trypsin with trypsin inhibitors, we showed that the protein binding capacity of methacrylate polymer elements and the sensitivity of MALDI-MS detection of proteins bound to these elements surpassed that of other 2- and 3-dimensional substrates tested Immobilized trypsin retained functional (enzymatic) activity within the protein microchip and the specificity of macromolecular interactions even in complex biological samples. We believe that the underlying technology should therefore be extensible to whole-proteome protein expression profiling and interaction mapping.     

C-reactive protein and C1q regulate platelet adhesion and activation on adsorbed immunoglobulin G and albumin

Blood platelets and C-reactive protein (CRP) are both used clinically as markers of ongoing inflammation, and both participate actively in inflammatory responses, although the biological effects are still incompletely understood. Rapidly adhering platelets express receptors for complement factor 1q (C1q) and the Fc part of immunoglobulin G (IgG), and CRP is known to activate/regulate complement via C1q binding, and to ligate FcgammaRs. In the present study, we used normal human IgG pre-adsorbed to a well-characterized methylated surface as a model solid-phase immune complex when investigating the effects of CRP and C1q on platelet adhesion and activation. Protein adsorption was characterized using ellipsometry and polyclonal antibodies, and human serum albumin (HSA) and non-coated surfaces were used as reference surfaces. Platelet adhesion to IgG and HSA was inhibited by both C1q and CRP. Furthermore, CRP (moderately) and C1q (markedly) decreased the spreading of adhering platelets. The combination of C1q and CRP was slightly more potent in reducing cell adhesion to IgG, and also impaired the adhesion to HSA and non-coated surfaces. Platelet production of thromboxane B2 (TXB(2)) was also reduced by C1q both in the presence and absence of CRP, whereas CRP alone had no effect on TXB(2) production. We conclude that CRP and C1q regulate the behaviour of platelets, and that this may be an important immunoregulatory mechanism during inflammatory conditions.     

Production and characterization of monoclonal antibodies against the hemolysin BL enterotoxin complex produced by Bacillus cereus.

A total of five hybridoma cell lines that produced monoclonal antibodies against the components of the hemolysin BL (HBL) enterotoxin complex and sphingomyelinase produced by Bacillus cereus were established and characterized. Monoclonal antibody 2A3 was specific for the B component, antibodies 1A12 and 8B12 were specific for the L(2) component, and antibody 1C2 was specific for the L(1) protein of the HBL enterotoxin complex. No cross-reactivity with other proteins produced by different strains of B. cereus was observed for monoclonal antibodies 2A3, 1A12, and 8B12, whereas antibody 1C2 cross-reacted with an uncharacterized protein of approximately 93 kDa and with a 39-kDa protein, which possibly represents one component of the nonhemolytic enterotoxin complex. Antibody 2A12 finally showed a distinct reactivity with B. cereus sphingomyelinase. The monoclonal antibodies developed in this study were also successfully applied in indirect enzyme immunoassays for the characterization of the enterotoxic activity of B. cereus strains. About 50% of the strains tested were capable of producing the HBL enterotoxin complex, and it could be demonstrated that all strains producing HBL were also highly cytotoxic.     

Synthetic peptide vaccine development: measurement of polyclonal antibody affinity and cross-reactivity using a new peptide capture and release system for surface plasmon resonance spectroscopy

A method has been developed for measurement of antibody affinity and cross-reactivity by surface plasmon resonance spectroscopy using the EK-coil heterodimeric coiled-coil peptide capture system. This system allows for reversible capture of synthetic peptide ligands on a biosensor chip surface, with the advantage that multiple antibody-antigen interactions can be analyzed using a single biosensor chip. This method has proven useful in the development of a synthetic peptide anti-Pseudomonas aeruginosa (PA) vaccine. Synthetic peptide ligands corresponding to the receptor binding domains of pilin from four strains of PA were conjugated to the E-coil strand of the heterodimeric coiled-coil domain and individually captured on the biosensor chip through dimerization with the immobilized K-coil strand. Polyclonal rabbit IgG raised against pilin epitopes was injected over the sensor chip surface for kinetic analysis of the antigen-antibody interaction. The kinetic rate constants, k(on) and k(off), and equilibrium association and dissociation constants, KA and KD, were calculated. Antibody affinities ranged from 1.14 x 10(-9) to 1.60 x 10(-5) M. The results suggest that the carrier protein and adjuvant used during immunization make a dramatic difference in antibody affinity and cross-reactivity. Antibodies raised against the PA strain K pilin epitope conjugated to keyhole limpet haemocyanin using Freund's adjuvant system were more broadly cross-reactive than antibodies raised against the same epitope conjugated to tetanus toxoid using Adjuvax adjuvant. The method described here is useful for detailed characterization of the interaction of polyclonal antibodies with a panel of synthetic peptide ligands with the objective of obtaining high affinity and cross-reactive antibodies in vaccine development.     

Highly efficient selection of phage antibodies mediated by display of antigen as Lpp-OmpA' fusions on live bacteria

Delayed infectivity panning (DIP) is a novel approach for the in vivo isolation of interacting protein pairs. DIP combines phage display and cell surface display of polypeptides as follows: an antigen is displayed in many copies on the surface of F(+) Escherichia coli cells by fusing it to a Lpp-OmpA' hybrid. To prevent premature, non-specific infection by phage, the cells are rendered functionally F(-) by growth at 16 degrees C. The antigen-displaying cells are used to capture antibody-displaying phage by virtue of the antibody-antigen interaction. Following removal of unbound phage, infection of the cells by bound phage is initiated by raising the temperature to 37 degrees C that facilitates F pilus expression. The phage then dissociate from the antigen and infect the bacteria through the F pilus. Using specific scFv antibodies and the human ErbB2 proto-oncogene and IL2-Ralpha chain as model antibody-antigen pairs, we demonstrate enrichment of those phage that display a specific antibody over phage that display an irrelevant antibody of over 1,000,000 in a single DIP cycle. We further show the successful isolation of anti-toxin, anti-receptor, anti-enzyme and anti-peptide antibodies from several immune phage libraries, a shuffled library and a large synthetic human library. The effectiveness of DIP makes it suitable for the isolation of rare clones present in large libraries.Since DIP can be applied for most of the phage libraries already existing, it could be a powerful tool for the rapid isolation and characterization of binders in numerous protein-protein interactions.     

RAGE binds C1q and enhances C1q-mediated phagocytosis

RAGE, the multiligand receptor of the immunoglobulin superfamily of cell surface molecules, is implicated in innate and adaptive immunity. Complement component C1q serves roles in complement activation and antibody-independent opsonization. Using soluble forms of RAGE (sRAGE) and RAGE-expressing cells, we determined that RAGE is a native C1q globular domain receptor. Direct C1q-sRAGE interaction was demonstrated with surface plasmon resonance (SPR), with minimum K(d) 5.6 μM, and stronger binding affinity seen in ELISA-like experiments involving multivalent binding. Pull-down experiments suggested formation of a receptor complex of RAGE and Mac-1 to further enhance affinity for C1q. C1q induced U937 cell adhesion and phagocytosis was inhibited by antibodies to RAGE or Mac-1. These data link C1q and RAGE to the recruitment of leukocytes and phagocytosis of C1q-coated material.     

Immunoglobulin M possesses two binding sites for complement subcomponent C1q, and soluble 1:1 and 2:1 complexes are formed in solution at reduced ionic strength

Soluble complexes were formed between C1q, a subunit of the first component of human complement, and four different Waldenstr?m IgM proteins at reduced ionic strengths. The equilibria between these complexes and the free proteins were studied in the ultracentrifuge. Complex formation was found to be a very sensitive function of the salt concentration, and at physiological ionic strength complex formation was negligible. The complexes were cross-linked with a water-soluble carbodiimide and separated by sucrose gradient centrifugation. Both 22 S 1:1 and 26 S 2:1 C1q X IgM complexes were formed; stoichiometry was established by cross-linking 125I-C1q with 131I-IgM and determining the ratios of the specific activities of the gradient-purified materials. The association process was studied as a function of protein concentration and was analyzed by Scatchard and Hill plots to yield stoichiometry, association constant, and degree of cooperativity. The results indicated that IgM has two identical and independent binding sites for C1q. The intrinsic association constant was found to vary between 10(6) M-1 at 0.084 M ionic strength to 10(4) M-1 at physiological ionic strength; the slope of the log-log plot gave a value of -6.0. The cross-linked complexes were examined by electron microscopy, and the C1q appeared to be attached to the IgM through the C1q heads, implying that the biologically significant binding sites were involved in this interaction. For the 2:1 complexes, the two C1q appeared to attach to opposite surfaces of the IgM, suggesting the presence of a pseudo-2-fold axis lying in the plane of the IgM disk.     

IgG subclass specificity to C1q determined by surface plasmon resonance using Protein L capture technique

Recombinant monoclonal antibodies (mAbs) have become an important category of biological therapeutics. mAbs share the same structures and biological functions as endogenous IgG molecules. One function is complement-dependent cytotoxicity (CDC) initiation by binding of C1q. Traditionally, ELISA methods have been utilized to measure C1q binding. A new robust capture method was established in this study to measure the binding affinity of C1q to antibodies by surface plasmon resonance (SPR). The utility of this method was demonstrated by determination of the difference in IgG subclass specificity of C1q binding.     

Production and characterization of a murine monoclonal IgM antibody to human C1q receptor (C1qR)

A hybridoma cell line that produces a monoclonal antibody (MAb) to cell surface C1q receptor (C1qR) has been produced by fusion of the P3 X 63-Ag8.653 mouse myeloma cell line with the spleen cells of a CD-1 mouse that had been hyperimmunized with viable Raji cell suspensions (5 X 10(7) cells/inoculum). This MAb, designated II1/D1, is an IgM antibody with lambda-light chain specificity. Radiolabeled or unlabeled, highly purified II1/D1 was used to determine that: this antibody competes for C1q binding sites on C1qR-bearing cells; the molecule recognized by this MAb is the C1qR; and cells that are known to bind C1q also bind II1/D1 in a specific manner. Western blot analysis of solubilized Raji, or U937 cell membranes, showed that the 125I-MAb detected a major protein band of approximately 85,000 m.w. in its unreduced state, indicating that the C1qR is similar, if not identical, in both types of cells. Analyses of 125I-II1/D1 binding experiments revealed that the antibody bound to Raji cells or U937 cells in a specific manner. Uptake of the antibody was saturable, with equilibrium virtually attained within 35 min. Scatchard analysis of the binding data using the intact MAb suggests that the affinity constant KD is 2.9 X 10(-10) M, and at apparent saturation, 24.6 ng of the antibody were bound per 2 X 10(6) cells, giving an estimated 7.8 X 10(3) antibody molecules bound per cell. That the II1/D1 antibody is specifically directed to the C1q was further evidenced by an ELISA in which the ability of C1qR-bearing cells to bind the MAb was abrogated by c-C1q in a specific and dose-dependent manner. These results indicate that the II1/D1 is a specific antibody directed against the C1q and can be a useful tool in studying the biologic interaction of human C1q with its receptors on a variety of cells.     

Monoclonal antibodies as probes of epithelial membrane polarization

Monoclonal antibodies directed against antigens in the apical plasma membrane of the toad kidney epithelial cell line A6 were produced to probe the phenomena that underlie the genesis and maintenance of epithelial polarity. Two of these antibodies, 17D7 and 18C3, were selected for detailed study here. 17D7 is directed against a 23-kD peptide found on both the apical and basolateral surfaces of the A6 epithelium whereas 18C3 recognizes a lipid localized to the apical membrane only. This novel observation of an apically localized epithelial lipid species indicates the existence of a specific sorting and insertion process for this, and perhaps other, epithelial plasma membrane lipids. The antibody-antigen complexes formed by both these monoclonal antibodies are rapidly internalized by the A6 cells, but only the 18C3-antigen complex is recycled to the plasma membrane. In contrast to the apical localization of the free antigen, however, the 18C3-antigen complex is recycled to both the apical and basolateral surface of the epithelium, which indicates that monoclonal antibody binding interferes in some way with the normal sorting process for this apical lipid antigen.     

Serum-dependent processing of late apoptotic cells for enhanced efferocytosis

Binding of the serum protein complement component C1q to the surface of dying cells facilitates their clearance by phagocytes in a process termed efferocytosis. Here, we investigate during which phase of apoptotic cell death progression C1q binding takes place. Purified C1q was found to bind to all dying cells and, albeit weaker, also to viable cells. The presence of serum abrogated completely the binding to viable cells. In addition, C1q binding to dying cells was limited to a specific subpopulation of late apoptotic/secondary necrotic cells. Co-culturing serum-treated apoptotic cells with human monocytes revealed a much higher phagocytosis of C1q-positive than of C1q-negative late apoptotic/secondary necrotic cells. But this phagocytosis-promoting activity could not be observed with purified C1q. Serum-treated C1q-positive late apoptotic/secondary necrotic cells exhibited a similar volume, a similar degraded protein composition, but a much lower DNA content in comparison with the remaining late apoptotic/secondary necrotic cells. This was mediated by a serum-bound nuclease activity that could be abrogated by G-actin, which is a specific inhibitor of serum DNase I. These results show that serum factors are involved in the prevention of C1q binding to viable cells and in the processing of late apoptotic/secondary necrotic cells promoting cell death progression toward apoptotic bodies. This process leads to the exposure of C1q-binding structures and facilitates efferocytosis.