Collagen Tail Research Articles

Limiting Role of Protein Disulfide Isomerase in the Expression of Collagen-tailed Acetylcholinesterase Forms in Muscle

The expression of acetylcholinesterase (AChE) in skeletal muscle is regulated by muscle activity; however, the underlying molecular mechanisms are incompletely understood. We show here that the expression of the synaptic collagen-tailed AChE form (ColQ-AChE) in quail muscle cultures can be regulated by muscle activity post-translationally. Inhibition of thiol oxidoreductase activity decreases expression of all active AChE forms. Likewise, primary quail myotubes transfected with protein disulfide isomerase (PDI) short hairpin RNAs showed a significant decrease of both the intracellular pool of all collagen-tailed AChE forms and cell surface AChE clusters. Conversely, overexpression of PDI, endoplasmic reticulum protein 72, or calnexin in muscle cells enhanced expression of all collagen-tailed AChE forms. Overexpression of PDI had the most dramatic effect with a 100% increase in the intracellular ColQ-AChE pool and cell surface enzyme activity. Moreover, the levels of PDI are regulated by muscle activity and correlate with the levels of ColQ-AChE and AChE tetramers. Finally, we demonstrate that PDI interacts directly with AChE intracellularly. These results show that collagen-tailed AChE form levels induced by muscle activity can be regulated by molecular chaperones and suggest that newly synthesized exportable proteins may compete for chaperone assistance during the folding process.

Evidence that a C1q/C1qR system regulates monocyte-derived dendritic cell differentiation at the interface of innate and acquired immunity

Growing evidence shows that C1q modulates the growth and function of cells committed to the monocyte-derived dendritic cell (DC) lineage. Because C1q regulates both innate and acquired immune responses, we postulated that C1q modulates the transition from monocytes to DCs, i.e. the interface between innate and acquired immunity. Human peripheral blood monocytes cultured with soluble C1q and DC growth factors (granulocyte-macrophage colony-stimulating factor + Interleukin-4) failed to down-regulate monocyte-associated (CD14, CD16) and up-regulate DC-associated (CD83, CD86) markers. Impaired DC differentiation was not due to apoptosis; further analysis revealed the development of CD14(hi)CD11c(hi)CD16 (+/-) cells that have previously been associated with both innate and acquired immunity. Monocyte-DC precursors expressed gC1qR, the receptor for globular heads of C1q, from the outset, while cC1qR, the receptor for the collagen tails of C1q, was expressed at low levels. Notably, the binding pattern of monoclonal antibodies specific to the globular heads of C1q indicated that C1q is bound to monocytes via globular heads, presumably through gC1qR. Moreover, gC1qR levels decreased, while cC1qR levels were dramatically amplified as monocytes differentiated into immature DC. Thus, specific C1q/C1q receptor (R) interactions may control the transition from the monocyte state (innate immunity) toward the professional antigen-presenting cell state (adaptive immunity).

Dissociation of Transcription, Translation, and Assembly of Collagen-tailed Acetylcholinesterase in Skeletal Muscle

The synaptic form of acetylcholinesterase (AChE) in skeletal muscle ColQ-AChE derives from two separate genes encoding the catalytic and the non-catalytic collagenic tail (ColQ) subunits, respectively. ColQ-AChE expression is regulated by muscle activity; however, how this regulation takes place in skeletal muscle remains poorly understood. In this study, we overexpressed or knocked down ColQ expression in skeletal muscle and found that the level of this non-catalytic component by itself was sufficient to change the levels of total AChE activity by promoting assembly of higher order oligomeric forms including the collagen-tailed forms. These results initially suggested that ColQ could be limiting in the assembly of synaptic ColQ-AChE during development and differentiation. We then determined the levels of ColQ protein and ColQ mRNA during primary quail muscle cell development and differentiation in culture (QMCs) and as a function of muscle activity. Surprisingly, we found dissociation between transcription and translation of the non-catalytic subunit from its assembly into ColQ-AChE. Furthermore, we found that the vast majority of the steady state ColQ molecules in mature quail muscle cultures are not assembled into ColQ-AChE, suggesting that they are either rapidly degraded or have alternative function(s).

Cholinesterases and the basal lamina at vertebrate neuromuscular junctions

Macromolecules of the cholinergic basal lamina are essential elements of the complex signaling processes governing development, function, and repair of the vertebrate neuromuscular junction. One special form of acetylcholinesterase (AChE) is anchored within BL through a collagen tail (ColQ) that binds heparan sulfate proteoglycans, such as perlecan, and the post-synaptic muscle specific kinase MuSK. New experimental approaches are probing the spatio-temporal dynamics of ColQ-AChE over days or weeks in vivo, thereby unraveling its interactions with other BL components, as well as pre-and post-synaptic elements. Concurrent advances in understanding of the biological effects of specific ColQ-AChE mutations prefigure improved diagnostics and clinical approaches for some congenital myasthenic syndromes.

Peripheral blood monocytes express surface C1q and its orientation may dictate early events of monocyte to dendritic cell differentiation (134.62)

Abstract C1q is a collagen-like glycoprotein comprising two functionally distinct regions–collagen tail (cC1q) and globular heads (gC1q). Our earlier experiments showed that C1q regulates monocyte to DC differentiation by suppressing DC development at the earliest stages. This suggested that C1q may be expressed on monocytes, and through interactions with C1qRs, could regulate early signals that lead to differentiation. Using ELISA and flow cytometric analysis, we confirmed the surface expression of C1q on fresh monocytes, and the level could be sustained beyond day 3 when monocytes were cultured with a mono-macrophage specific growth factor (M-CSF). In the presence of DC growth factors (GM-CSF+IL-4) however, surface expression rapidly declined coinciding with firm commitment to the DC lineage on day 3. Anti-gC1q IgG bound only to macrophages but not to monocytes or immature DCs, suggesting that C1q is bound to monocytes via gC1q, whereas on macrophages it is anchored via cC1q. Thus the orientation of surface C1q may dictate regulation of the early processes that signal progression from the monocyte lineage (innate) towards the DC lineage (adaptive immunity). More importantly, since M-CSF is a normal constituent in the blood, the expression of C1q on fresh monocytes may reflect an M-CSF/C1q coupled regulatory process operating to maintain steady state. [Supported by NIH Grant R01 AI-060866 (to BG) and R03 AR-05396 (to FSS)]

C1q‐ and Collectin‐Dependent Phagocytosis of Apoptotic Host Cells by the Intestinal ProtozoanEntamoeba histolytica

Entamoeba histolytica, the cause of invasive amebiasis, phagocytoses apoptotic host cells during tissue invasion. In mammals, collectin family members (e.g., mannose-binding lectin [MBL]) and the structurally related protein C1q bind to apoptotic cells and stimulate macrophage phagocytosis via a conserved collagenous tail domain. The collectins also bind to bacteria, the usual source of nutrients for E. histolytica. To test the possibility that the collectins are ligands that stimulate E. histolytica phagocytosis, we used a flow cytometry-based assay for amebic phagocytosis, a method for making single-ligand particles to delineate a given ligand's ability to initiate phagocytosis, and purified human C1q, MBL, and collagenous collectin tails. Apoptotic lymphocytes opsonized with serum or human C1q were phagocytosed more efficiently than control cells, an effect that was dependent on ligand density. C1q and the collectins alone were adequate to trigger amebic phagocytosis, because single-ligand particles coated with C1q, MBL, or purified collectin tails were phagocytosed more efficiently than control particles. Furthermore, C1q, MBL, and the tail domain of C1q were all chemoattractants for E. histolytica. C1q and MBL can serve as opsonins on apoptotic cells that stimulate E. histolytica phagocytosis, an effect mediated at least in part by the collagenous collectin tail domain.

Transcriptional control of different subunits of AChE in muscles: Signals triggered by the motor nerve-derived factors

Acetylcholinesterase (AChE) is a highly polymorphic enzyme. Alternative splicing in the 3′ region of the primary transcript generates different subunits that contain the same catalytic domain but with distinct carboxyl termini. In mammals, the AChE R variant produces a soluble monomer that is up-regulated in the brain during stress. The AChE H variant produces a GPI-anchored dimer that is mainly expressed in blood cells, while AChE T variant is largely predominant in the brain and muscle. AChE T subunits associate with a collagen tail subunit (ColQ) forming asymmetric AChE species (A 4, A 8, and A 12 AChE) in muscle, and also form amphiphilic tetramers associated with a proline-rich membrane anchor (PRiMA) as globular AChE (G 4 AChE) in brain and muscle. The formation of these AChE forms depends on the physiological status of the muscles, and on the innervating nerves. The motor nerves achieve this regulation by two distinct mechanisms: release of the trophic factor calcitonin gene-related peptide (CGRP) and nerve-evoked electrical activity, which differentially regulate the expression levels of AChE T, PRiMA and ColQ via different downstream signaling cascades. The regulatory mechanisms provided by the nerve are important to account for the different expression patterns of AChE and associated proteins in fast- and slow-twitch muscles.

Lamellipodin proline rich peptides associated with native plasma butyrylcholinesterase tetramers

BChE (butyrylcholinesterase) protects the cholinergic nervous system from organophosphorus nerve agents by scavenging these toxins. Recombinant human BChE produced from transgenic goat to treat nerve agent intoxication is currently under development. The therapeutic potential of BChE relies on its ability to stay in the circulation for a prolonged period, which in turn depends on maintaining tetrameric quaternary configuration. Native human plasma BChE consists of 98% tetramers and has a half-life (t((1/2))) of 11-14 days. BChE in the neuromuscular junctions and the central nervous system is anchored to membranes through interactions with ColQ (AChE-associated collagen tail protein) and PRiMA (proline-rich membrane anchor) proteins containing proline-rich domains. BChE prepared in cell culture is primarily monomeric, unless expressed in the presence of proline-rich peptides. We hypothesized that a poly-proline peptide is an intrinsic component of soluble plasma BChE tetramers, just as it is for membrane-bound BChE. We found that a series of proline-rich peptides was released from denatured human and horse plasma BChE. Eight peptides, with masses from 2072 to 2878 Da, were purified by HPLC and sequenced by electrospray ionization tandem MS and Edman degradation. All peptides derived from the same proline-rich core sequence PSPPLPPPPPPPPPPPPPPPPPPPPLP (mass 2663 Da) but varied in length at their N- and C-termini. The source of these peptides was identified through database searching as RAPH1 [Ras-associated and PH domains (pleckstrin homology domains)-containing protein 1; lamellipodin, gi:82581557]. A proline-rich peptide of 17 amino acids derived from lamellipodin drove the assembly of human BChE secreted from CHO (Chinese-hamster ovary) cells into tetramers. We propose that the proline-rich peptides organize the 4 subunits of BChE into a 340 kDa tetramer, by interacting with the C-terminal BChE tetramerization domain.

Identification of C1q receptor associated surface molecules on the monocyte‐like cell line U937

The C1q receptor (C1qR) comprises of at least two unrelated surface proteins, which bind C1q via either its globular heads (gC1qR) or collagen tail (cC1qR). Although these non‐transmembrane proteins bind C1q and induce biological responses, the mechanism by which they do so is incompletely understood. The present studies were undertaken to identify potential new receptors or associated molecules that may have eluded identification due to limitations of previous techniques. Therefore, we used solubilized membrane proteins from U937 cells (total 2×108 cells) that were first surface biotinylated by a method that targeted either the amino groups or the carbohydrate moieties of proteins. Then, immunocapture ELISA was used to confirm that the known receptors—gC1qR and cC1qR—are surface labeled. Next, we used anti‐gC1qR mAb 74.5.2 to identify membrane proteins that are co‐captured with gC1qR. The results showed the presence of CD44 and β1 integrin. Furthermore, when membrane proteins were incubated with C1q coated wells, both CD44 and β1 integrin were bound to C1q in addition to the expected gC1qR and cC1qR. These results indicate that C1qR probably consists of transmembrane (CD44 and β1 integrin) and non‐transmembrane (gC1qR and cC1qR) surface proteins that together forge a docking/signaling partnership. [Supported by NIH‐NIAID Grant R01 AI‐060866]

C1q is a molecular switch dictating the monocyte to dendritic cell (DC) transition and arrests DCs in an immature phenotype

Complement protein C1q plays a crucial role in regulating peripheral tolerance by facilitating clearance of apoptotic debris and modulating T cell proliferation. Moreover, C1q is emerging as a moderator of dendritic cell (DC) activity. Immature DCs (iDC) are able to secrete C1q. We postulated that such locally secreted C1q regulates the transition from monocytes to DCs. Using flow cytometric analysis we show here that monocyte‐derived DCs cultured in the presence of soluble C1q failed to down regulate CD14 and expressed reduced levels of CD83, DR and CD86. The inhibition of DC maturation was not due to apoptosis, as assessed by lack of annexin V binding and morphological examination. Instead, multiparametric analysis revealed the development of CD14+CD11c+CD16+/− cells representing a subset of iDCs. Monocyte‐DC precursors from the outset expressed gC1qR, the receptor for globular heads of C1q. In contrast, cC1qR, the receptor for the collagen tails of C1q, was lacking on these cells despite the expression of its partner CD91, indicating that inhibition of DC growth by C1q occurs via C1q‐gC1qR interactions. Significantly, pathogens such as hepatitis C virus, which bind gC1qR, may exploit this system by mimicking C1q to escape the acquired immune response. We propose that C1q operates at a critical checkpoint during the monocyte to DC transition and helps maintain immune tolerance by signaling via gC1qR. [Supported by NIH Grant R01 AI‐060866 (to BG) and R03 AR‐05396 (to FSS)]

Acetylcholinesterase from the invertebrate Ciona intestinalis is capable of assembling into asymmetric forms when co-expressed with vertebrate collagenic tail peptide

To learn more about the evolution of the cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase in the vertebrates, we investigated the AChE activity of a deuterostome invertebrate, the urochordate Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for the enzyme in COS-7 cells. Evidence from kinetics, pharmacology, molecular biology, and molecular modeling confirms that the enzyme is AChE. Sequence analysis and molecular modeling also indicate that the cDNA codes for the AChE(T) subunit, which should be able to produce all three globular forms of AChE: monomers (G(1)), dimers (G(2)), and tetramers (G(4)), and assemble into asymmetric forms in association with the collagenic subunit collagen Q. Using velocity sedimentation on sucrose gradients, we found that all three of the globular forms are either expressed in cells or secreted into the medium. In cell extracts, amphiphilic monomers (G(1)(a)) and non-amphiphilic tetramers (G(4)(na)) are found. Amphiphilic dimers (G(2)(a)) and non-amphiphilic tetramers (G(4)(na)) are secreted into the medium. Co-expression of the catalytic subunit with Rattus norvegicus collagen Q produces the asymmetric A(12) form of the enzyme. Collagenase digestion of the A(12) AChE produces a lytic G(4) form. Notably, only globular forms are present in vivo. This is the first demonstration that an invertebrate AChE is capable of assembling into asymmetric forms. We also performed a phylogenetic analysis of the sequence. We discuss the relevance of our results with respect to the evolution of the ChEs in general, in deuterostome invertebrates, and in chordates including vertebrates.

The Scl1 protein of M6‐type group A Streptococcus binds the human complement regulatory protein, factor H, and inhibits the alternative pathway of complement

Non-specific activation of the complement system is regulated by the plasma glycoprotein factor H (FH). Bacteria can avoid complement-mediated opsonization and phagocytosis through acquiring FH to the cell surface. Here, we characterize an interaction between the streptococcal collagen-like protein Scl1.6 of M6-type group A Streptococcus (GAS) and FH. Using affinity chromatography with immobilized recombinant Scl1.6 protein, we co-eluted human plasma proteins with molecular weight of 155 kDa, 43 kDa and 38 kDa. Mass spectrometry identified the 155 kDa band as FH and two other bands as isoforms of the FH-related protein-1. The identities of all three bands were confirmed by Western immunoblotting with specific antibodies. Structure-function relation studies determined that the globular domain of the Scl1.6 variant specifically binds FH while fused to collagenous tails of various lengths. This binding is not restricted to Scl1.6 as the phylogenetically linked Scl1.55 variant also binds FH. Functional analyses demonstrated the cofactor activity of the rScl1.6-bound FH for factor I-mediated cleavage of C3b. Finally, purified FH bound to the Scl1.6 protein present in the cell wall material obtained from M6-type GAS. In conclusion, we have identified a functional interaction between Scl1 and plasma FH, which may contribute to GAS evasion of complement-mediated opsonization and phagocytosis.

Novel COLQ mutation 950delC in synaptic congenital myasthenic syndrome and symptomatic heterozygous relatives

The synaptic form of congenital myasthenic syndrome (CMS) is a rare autosomal recessive disease affecting neuromuscular transmission. Mutations in the COLQ gene that encodes the collagenic tail subunit ( ColQ) of asymmetric acetylcholinesterase lead to endplate acetylcholinesterase deficiency. We report two children suffering from synaptic CMS due to two compound heterozygous COLQ mutations, IVS1-1G > A and a novel mutation, 950delC. Furthermore, we found familial occurrence of congenital ptosis in heterozygous carriers of 950delC, mimicking a dominant negative effect. Considering the lack of a clear genotype–phenotype-relation in synaptic CMS, several authors speculated on the influence of additional modifying factors. Consequently, involvement of such factors in this report of familial congenital ptosis cannot be excluded.

Atomistic Simulation Study of Calcium, Phosphate and Fluoride Ion Association to the Teleopeptide‐Tails of Collagen – Initial Steps to Biomineral Formation

AbstractThe attachment of single ions to putative adsorption sites in the tails of collagen fibers is investigated by means of molecular dynamics simulations and discussed with respect to the very early steps of apatite/collagen biomineral formation. Our studies clearly demonstrate an increased flexibility of the tails of the triple‐helical collagen protein. Apart from the termini of the backbone, several side chains were also observed to be freely accessible to ion attachment from aqueous solution. The teleopeptide was systematically scanned for suitable adsorption sites for calcium, phosphate and fluoride ions. Association of these ions was then explored from potential of mean force calculations. The resulting energy profiles reveal a variety of favorable protein‐ion bonds and hint at the suitability of the collagen tails to promote apatite aggregation.

The “wisdom” of lung surfactant: balancing host defense and surface tension-reducing functions

the mixture of lipids and proteins that comprises pulmonary surfactant is both complex and multifunctional. Although surfactant was originally identified as a lipoprotein complex that reduces surface tension at the air-liquid interface of the lung ([4][1], [20][2]), both in vitro and in vivo studies

Conformational Features of a Natural Break in the Type IV Collagen Gly-X-Y Repeat

Fibrillar collagens have an absolute requirement for Gly as every 3rd residue, whereas breaks in the Gly-X-Y repeating pattern are found normally in the triple helix domains of non-fibrillar collagens, such as type IV collagen in basement membranes. In this study, a model 30-mer peptide is designed to include the interruption GPOGAAVMGPOGPO found in the alpha5 chain of type IV collagen. The GAAVM peptide forms a stable triple helix, with Tm= 29 degrees C. When compared with a control peptide with Gly as every 3rd residue, the GAAVM peptide has a marked decrease in the 225 nm maximum of its CD spectrum and a 10 degrees C drop in stability. A 50% decrease in calorimetric enthalpy is observed, which may result from disruption of ordered water structure anchored by regularly placed backbone carbonyls. NMR studies on specific 15N-labeled residues within the GAAVM peptide indicate a normal triple helical structure for Gly-Pro-Hyp residues flanking the break. The sequence within the break is not disordered but shows altered hydrogen exchange rates and an abnormal Val chemical shift. It was previously reported that a peptide designed to model a similar kind of interruption in the peptide (Pro-Hyp-Gly)10, (GPOGPOPOGPO), is unable to form a stable triple helix, and replacement of GAA by GPO or VM by PO within the GAAVM break decreases the stability. Thus, rigid imino acids are unfavorable within a break, despite their favorable stabilization of the triple helix itself. These results suggest some non-random structure typical of this category of breaks in the Gly-X-Y repeat of the triple helix.

Targeting acetylcholinesterase to the neuromuscular synapse

The collagen-tailed form of acetylcholinesterase (ColQ-AChE) is the major if not unique form of the enzyme associated with the specialized synaptic basal lamina at the neuromuscular junction (NMJ). This enzyme form consists of both catalytic and non-catalytic subunits encoded by separate genes, assembled as three enzymatic tetramers attached to the three-stranded collagen-like tail. We have previously shown that catalytic subunits are assembled in the rough endoplasmic reticulum and that after approximately 90 min a subset of these tetramers assemble with collagenic tail subunits in the Golgi apparatus. In muscle, blocking ER to Golgi transport with Brefeldin A prevents the appearance of ColQ-AChE, consistent with assembly of asymmetric forms in the Golgi. Moreover, newly synthesized and assembled ColQ-AChE associates with perlecan intracellularly and can only be co-immunoprecipitated with anti-perlecan antibodies 90 min after the first appearance of catalytic subunits. Once assembled, the ColQ-AChE/perlecan complex is externalized where it co-localizes with other components of the NMJ including dystroglycan, rapsyn, laminin and MuSK. These clusters tend to form over the nuclei that are expressing the components, suggesting local vectorial transport to the cell surface, and may form a primary scaffold that in turn can capture other molecular constituents of the neuromuscular synapse. While most AChE clusters on quail myotubes are devoid of acetylcholine receptors, treatment of the culture with recombinant agrin results in a rapid translocation of receptors to the AChE clusters in less than 4 h. It remains to be determined if MuSK is localized to the clusters. In vivo, AChE transcripts and enzyme are more highly expressed at the NMJs, implying higher rates of AChE translation and assembly in the synaptic regions, and hence more ColQ-AChE for localized export. We have previously shown that binding sites for ColQ-AChE are concentrated at sites of nerve-muscle contact where they colocalize with AChR and perlecan. ColQ-AChE binds directly to perlecan using solid phase microtiter plate assay, the Biacore assay, and co-immunoprecipitations. Moreover, perlecan binds to dystroglycan at the NMJ. In perlecan or dystroglycan null mice there is no accumulation of AChE at the NMJ, supporting the hypothesis that this heparan sulfate proteoglycan is an essential component of the ColQ-AChE localization mechanism. Together, these studies suggest a model of synaptic development whereby AChE can be targeted to and clustered on the muscle membrane together with dystroglycan and perlecan to form scaffolds to which AChR can be clustered through activation of the MuSK receptor. At mature synapses ColQ-AChE is secreted directly into the synaptic cleft where it binds to the heparan sulfate proteoglycan perlecan as well as potentially other molecules including MuSK, as was recently reported.

Congenital endplate acetylcholinesterase deficiency responsive to ephedrine

The authors describe two patients with congenital myasthenic syndrome (CMS) with end plate acetylcholinesterase (AChE) deficiency caused by mutations in the collagenic tail (ColQ) of AChE: a homozygous C-terminal Y230S mutation in Patient 1 and Y430S and a C-terminal splice-site mutation in Patient 2. In Patient 1, a Prostigmin (neostigmine bromide) test failed to distinguish between AChE deficiency and a slow-channel CMS. Both patients responded dramatically to ephedrine therapy.

Congenital myasthenic syndromes.

Congenital myasthenic syndromes are a heterogeneous group of diseases caused by genetic defects affecting neuromuscular transmission. In this article, a strategy that leads to the diagnosis of congenital myasthenic syndromes is presented, and recent advances in the clinical, genetic and molecular aspects of congenital myasthenic syndrome are outlined. Besides the identification of new mutations in genes already known to be implicated in congenital myasthenic syndromes (genes for the acetylcholine receptor subunits and the collagen tail of acetylcholinesterase), mutations in other genes have more recently been discovered and characterized (genes for choline acetyltransferase, rapsyn, and the muscle sodium channel SCN4A). Fluoxetine has recently been proposed as an alternative treatment for 'slow channel' congenital myasthenic syndrome. The characterization of congenital myasthenic syndromes comprises two complementary steps: establishing the diagnosis and identifying the pathophysiological type of congenital myasthenic syndrome. Characterization of the type of congenital myasthenic syndrome has allowed it to be classified as caused by presynaptic, synaptic and postsynaptic defects. A clinically and muscle histopathologically oriented genetic study has identified several genes in which mutations cause the disease. Despite comprehensive characterization, the phenotypic expression of one given gene involved is variable, and the aetiology of many congenital myasthenic syndromes remains to be discovered.

The C4b-binding Protein-Protein S Complex Inhibits the Phagocytosis of Apoptotic Cells

The phagocytosis of apoptotic cells is a complex process involving numerous interactions between the target cell and the macrophage. We have examined a role of the major soluble inhibitor of the classic and lectin complement pathways, C4b-binding protein (C4BP), in the clearance of apoptotic cells. The major form of C4BP present in blood is composed of seven alpha-chains and one beta-chain, which binds protein S (PS). Approximately 70% of all PS in human plasma is trapped in such a complex and is able to localize C4BP to the surface of apoptotic cells due to the high affinity to phosphatidylserine. Free PS has recently been shown to enhance phagocytosis of apoptotic cells by macrophages. We observed a stimulatory effect of free PS on the engulfment of apoptotic cells (BL-41 and Jurkat) by primary human macrophages or THP-1 cells and a decrease of activity in serum depleted of PS in agreement with previous results. However, we also show that the process is strongly inhibited in the presence of the C4BP-PS complex. Addition of the C4BP-PS complex to serum deficient in both molecules abolished the enhancing effect of serum on phagocytosis. The effect of both free PS and the C4BP-PS complex could be inhibited with monoclonal antibody directed against the Gla domain of PS. Although the presence of the C4BP-PS complex on apoptotic cells may lead to decreased phagocytosis, it may still be beneficial to the host, since it could prevent secondary necrosis because it inhibits further complement attack.