Ligand iC3b Research Articles

Microglial CR3 promotes neuron ferroptosis via NOX2-mediated iron deposition in rotenone-induced experimental models of Parkinson's disease

The activation of complement receptor 3 (CR3) in microglia contributes to neurodegeneration in neurological disorders, including Parkinson's disease (PD). However, it remains unclear for mechanistic knowledge on how CR3 mediates neuronal damage. In this study, the expression of CR3 and its ligands iC3b and ICAM-1 was found to be up-regulated in the midbrain of rotenone PD mice, which was associated with elevation of iron content and disruption of balance of iron metabolism proteins. Interestingly, genetic deletion of CR3 blunted iron accumulation and recovered the expression of iron metabolism markers in response to rotenone. Furthermore, reduced lipid peroxidation, ferroptosis of dopaminergic neurons and neuroinflammation were detected in rotenone-lesioned CR3−/− mice compared with WT mice. The regulatory effect of CR3 on ferroptotic death of dopaminergic neurons was also mirrored in vitro. Mechanistic study revealed that iron accumulation in neuron but not the physiological contact between microglia and neurons was essential for microglial CR3-regulated neuronal ferroptosis. In a cell-culture system, microglial CR3 silence significantly dampened iron deposition in neuron in response to rotenone, which was accompanied by mitigated lipid peroxidation and neurodegeneration. Furthermore, ROS released from activated microglia via NOX2 was identified to couple microglial CR3-mediated iron accumulation and subsequent neuronal ferroptosis. Finally, supplementation with exogenous iron was found to recover the sensitivity of CR3−/− mice to rotenone-induced neuronal ferroptosis. Altogether, our findings suggested that microglial CR3 regulates neuron ferroptosis through NOX2 -mediated iron accumulation in experimental Parkinsonism, providing novel points of the immunopathogenesis of neurological disorders.

Dysfunction of complement receptors CR3 (CD11b/18) and CR4 (CD11c/18) in pre-eclampsia: a genetic and functional study.

To study genetic variants and their function within genes coding for complement receptors in pre-eclampsia. A case-control study. Pre-eclampsia is a common vascular disease of pregnancy. The clearance of placenta-derived material is one of the functions of the complement system in pregnancy. We genotyped 500 women with pre-eclamptic pregnancies and 190 pregnant women without pre-eclampsia, as controls, from the FINNPEC cohort, and 122 women with pre-eclamptic pregnancies and 1905 controls from the national FINRISK cohort. The functional consequences of genotypes discovered by targeted exomic sequencing were explored by analysing the binding of the main ligand iC3b to mutated CR3 or CR4, which were transiently expressed on the surface of COS-1 cells. Allele frequencies were compared between pre-eclamptic pregnancies and controls in genetic studies. The functional consequences of selected variants were measured by binding assays. The most significantly pre-eclampsia-linked CR3 variant M441K (P=4.27E-4, OR=1.401, 95%CI=1.167-1.682) displayed a trend of increased adhesion to iC3b (P=0.051). The CR4 variant A251T was found to enhance the adhesion of CR4 to iC3b, whereas W48R resulted in a decrease of the binding of CR4 to iC3b. Results suggest that changes in complement-facilitated phagocytosis are associated with pre-eclampsia. Further studies are needed to ascertain whether aberrant CR3 and CR4 activity leads to altered pro- and anti-inflammatory cytokine responses in individuals carrying the associated variants, and the role of these receptors in pre-eclampsia pathogenesis. Genetic variants of complement receptors CR3 and CR4 have functional consequences that are associated with pre-eclampsia.

Role of the β-Subunit Arginine/Lysine Finger in Integrin Heterodimer Formation and Function

Formation of the integrin alphabeta heterodimer is essential for cell surface expression and function. At the core of the alphabeta interface is a conserved Arg/Lys "finger" from the beta-subunit that inserts into a cup-like "cage" formed of two layers of aromatic residues in the alpha-subunit. We evaluated the role of this residue in heterodimer formation in an alphaA-lacking and an alphaA-containing integrin alphaVbeta3 and alphaMbeta2 (CD11b/CD18), respectively. Arg261 of beta3 was mutated to Ala or Glu; the corresponding Lys252 of beta2 was mutated to Ala, Arg, Glu, Asp, or Phe; and the effects on heterodimer formation in each integrin examined by ELISA and immunoprecipitation in HEK 293 cells cotransfected with plasmids encoding the alpha- and beta-subunits. The Arg261Glu (but not Arg261Ala) substitution significantly impaired cell surface expression and heterodimer formation of alphaVbeta3. Although Lys252Arg, and to a lesser extent Lys252Ala, were well tolerated, each of the remaining substitutions markedly reduced cell surface expression and heterodimer formation of CD11b/CD18. Lys252Arg and Lys252Ala integrin heterodimers displayed a significant increase in binding to the physiologic ligand iC3b. These data demonstrate an important role of the Arg/Lys finger in formation of a stable integrin heterodimer, and suggest that subtle changes at this residue affect the activation state of the integrin.

Investigation of the mechanisms of Ultraviolet B-induced immunosuppression

中波紫外线是一种到达地球表面最具有生物活性的紫外线,是光生物学研究的主要对象.概述去氧核糖核酸、尿刊酸、活性氧系列、细胞因子及调节性T细胞方面的研究,尤其是重点讨论补体在中波紫外线导致免疫抑制方面的研究.在中波紫外线照射皮肤后,可使真皮内补体C3衍生为iC3b,,同时在一些因子作用下,血中单核细胞游移至真皮和表皮.它们表面的受体CR3和iC3b配体作用可上调白介素-10,而下调白介素-12,这一调节作用又与分裂原活化蛋白激酶,特别是对提高细胞外信号调节激酶相关。

The β-tail domain (βTD) regulates physiologic ligand binding to integrin CD11b/CD18

AbstractCrystallographic and electron microscopy studies revealed genuflexed (bent) integrins in both unliganded (inactive) and physiologic ligandbound (active) states, suggesting that local conformational changes are sufficient for activation. Herein we have explored the role of local changes in the contact region between the membrane-proximal β-tail domain (βTD) and the ligand-binding βA domain of the bent conformation in regulating interaction of integrin CD11b/CD18 (αMβ2) with its physiologic ligand iC3b. We replaced the βTD CD loop residues D658GMD of the CD18 (β2) subunit with the equivalent D672SSG of the β3 subunit, with AGAA or with NGTD, expressed the respective heterodimeric receptors either transiently in epithelial HEK293T cells or stably in leukocytes (K562), and measured their ability to bind iC3b and to conformation-sensitive mAbs. In the presence of the physiologic divalent cations Ca2+ plus Mg2+ (at 1 mM each), the modified integrins showed increased (in HEK293) or constitutive (in K562) binding to iC3b compared with wild-type receptors. K562 expressing the βTD-modified integrins bound in Ca2+Mg2+ to the βA-directed high-affinity reporter mAb 24 but not to mAb KIM127, a reporter of the genu-straightened state. These data identify a role for the membrane proximal βTD as an allosteric modulator of integrin activation.

Inhibition of monocyte‐derived dendritic cell differentiation and interleukin‐12 production by complement iC3b via a mitogen‐activated protein kinase signalling pathway

We have previously demonstrated that iC3b is deposited at the dermal-epidermal junction of the skin following ultraviolet (UV) exposure and that it plays a role in UV-induced immunosuppression and antigenic tolerance. In vitro, iC3b differentially regulates monocyte production of interleukin-10 (IL-10) and IL-12. Additionally, iC3b arrests monocytic cell differentiation into CD1c-expressing dendritic cell (DC) precursors. The present study addresses mitogen-activated protein kinase (MAPK) signalling following the cross-linking of CR3 by its ligand iC3b with regard to monocyte differentiation and cytokine regulation. Sheep erythrocytes were coated with IgM alone (EA) or iC3b (EAiC3b) to allow for CR3 cross-linking onto monocytes. EAiC3b increased the phosphorylation (p) of extracellular signal-regulated kinase (ERK) MAPK in fresh human monocyte, particularly in monocyte-derived DC (MDDC) that were differentiated by means of GM-CSF (1000 U/ml) and IL-4 (200 U/ml) for 2 days before iC3b exposure for an additional 24 h (P=0.034, n=3). CD1a expression, induced by GM-CSF and IL-4, was inhibited by iC3b (EAiC3b vs. EA, P=0.012, n=4). Conversely, the inhibition of ERK by the specific inhibitor (PD98059), but not the p-38 inhibitor SB203580, restored CD1a expression (P=0.011, n=4) in iC3b-stimulated MDDC. Concordantly, the inhibition of ERK during iC3b exposure fully reversed the inhibition of IL-12p70 induction in MDDC by 95% (P<0.01, n=4) and decreased IL-10 production. Taken together, our data demonstrate that iC3b interferes with MDDC differentiation and IL-12 and IL-10 production is mediated via an ERK MAPK-dependent mechanism. Thus, ERK MAPK inhibition may represent a therapeutic strategy for preventing monocytic precursor diversion away from DC differentiation when monocytes enter injured tissues in which iC3b is generated, such as UV-exposed skin.

Kinetic analysis of the interactions of complement receptor 2 (CR2, CD21) with its ligands C3d, iC3b, and the EBV glycoprotein gp350/220.

The molecular mechanisms involved in the interaction of complement receptor 2 (CR2) with its natural ligands iC3b and C3d are still not well understood. In addition, studies regarding the binding site(s) of the receptor on C3 as well as the affinities of the C3 fragments for CR2 have produced contradictory results. In the present study, we have used surface plasmon resonance technology to study the interaction of CR2 with its ligands C3d, iC3b, and the EBV surface glycoprotein gp350/220. We measured the kinetics of binding of the receptor to its ligands, examined the influence of ionic contacts on these interactions, and assessed whether immobilized and soluble iC3b bound with similar kinetics to CR2. Our results indicate that 1) gp350 binding to CR2 follows a simple 1:1 interaction, whereas that of the C3 fragments is more complex and involves more than one intramolecular component; 2) kinetic differences exist between the binding of C3d and iC3b to CR2, which may be due to an additional binding site found on the C3c region of iC3b; and 3) iC3b binds to CR2 with different kinetics, depending on whether the iC3b is in solution or immobilized on the surface. These findings suggest that binding of CR2 to iC3b and C3d is more complex than previously thought.

Structure-guided identification of C3d residues essential for its binding to complement receptor 2 (CD21).

A vital role for complement in adaptive humoral immunity is now beyond dispute. The crucial interaction is that between B cell and follicular dendritic cell-resident complement receptor 2 (CR2, CD21) and its Ag-associated ligands iC3b and C3dg, where the latter have been deposited as a result of classical pathway activation. Despite the obvious importance of this interaction, the location of a CR2 binding site within C3d, a proteolytic limit fragment of C3dg retaining CR2 binding activity, has not been firmly established. The recently determined x-ray structure of human C3d suggested a candidate site that was remote from the site of covalent attachment to Ag and consisted of an acidic residue-lined depression, which accordingly displays a significant electronegative surface potential. These attributes were consistent with the known ionic strength dependence of the CR2-C3d interaction and with the fact that a significant electropositive surface was apparent in a modeled structure of the C3d-binding domains of CR2. Therefore, we have performed an alanine scan of all of the residues within and immediately adjacent to the acidic pocket in C3d. By testing the mutant iC3b molecules for their ability to bind CR2, we have identified two separate clusters of residues on opposite sides of the acidic pocket, specifically E37/E39 and E160/D163/I164/E166, as being important CR2-contacting residues in C3d. Within the second cluster even single mutations cause near total loss of CR2 binding activity. Consistent with the proposed oppositely charged nature of the interface, we have also found that removal of a positive charge immediately adjacent to the acidic pocket (mutant K162A) results in a 2-fold enhancement in CR2 binding activity.

Computational design of an integrin I domain stabilized in the open high affinity conformation.

We have taken a computational approach to design mutations that stabilize a large protein domain of approximately 200 residues in two alternative conformations. Mutations in the hydrophobic core of the alphaMbeta2 integrin I domain were designed to stabilize the crystallographically defined open or closed conformers. When expressed on the cell surface as part of the intact heterodimeric receptor, binding of the designed open and closed I domains to the ligand iC3b, a form of the complement component C3, was either increased or decreased, respectively, compared to wild type. Moreover, when expressed in isolation from other integrin domains using an artificial transmembrane domain, designed open I domains were active in ligand binding, whereas designed closed and wild type I domains were inactive. Comparison to a human expert designed open mutant showed that the computationally designed mutants are far more active. Thus, computational design can be used to stabilize a molecule in a desired conformation, and conformational change in the I domain is physiologically relevant to regulation of ligand binding.

Folding and Function of I Domain-deleted Mac-1 and Lymphocyte Function-associated Antigen-1

In those integrins that contain it, the I domain is a major ligand recognition site. The I domain is inserted between beta-sheets 2 and 3 of the predicted beta-propeller domain of the integrin alpha subunit. We deleted the I domain from the integrin alpha(M) and alpha(L) subunits to give I-less Mac-1 and lymphocyte function-associated antigen-1 (LFA-1), respectively. The I-less alpha(M) and alpha(L) subunits were expressed in association with the wild-type beta(2) subunit on the surface of transfected cells and bound to all the monoclonal antibodies mapped to the putative beta-propeller and C-terminal regions of the alpha(M) and alpha(L) subunits, suggesting that the folding of these domains is independent of the I domain. I-less Mac-1 bound to the ligands iC3b and factor X, but this binding was reduced compared with wild-type Mac-1. In contrast, I-less Mac-1 did not bind to fibrinogen or denatured bovine serum albumin. Binding to iC3b and factor X by I-less Mac-1 was inhibited by the function-blocking antibody CBRM1/32, which binds to the beta-propeller domain of the alpha(M) subunit. I-less LFA-1 did not bind its ligands intercellular adhesion molecule-1 and -3. Thus, the I domain is not essential for the folding, heterodimer formation, and surface expression of Mac-1 and LFA-1 and is required for binding to some ligands, but not others.

Role of beta2 integrins in the prevention of apoptosis induction in chronic lymphocytic leukemia B cells.

Immunologically committed lymphocytes, especially mature, leukemic B cells, proliferate then accumulate without further cell division in chronic lymphocytic leukemia patients (CLL). These mature, leukemic B cells often produce autoantibodies. Under normal circumstances, immunologically committed lymphocytes that are autoreactive are deleted by a programmed cell death mechanism. In CLL cells, these mechanisms appear to be inhibited; therefore, cells accumulate rather than be destroyed. To understand the mechanism by which cell survival is selected over death in CLL cells, we studied the role of beta2 integrins and their ligands in the regulation of apoptosis. CLL cells were treated with monoclonal antibodies directed against beta2 integrins. Antibodies directed against the I-domain of the alpha chain of CD11b/CD18 inhibited apoptosis. The identity of the physiological ligand or counter-receptor for beta2 integrins that was required for the inhibition of apoptosis induction was sought. The ligand iC3b, but not ICAM-1 or fibrinogen, was identified as a ligand that could prevent apoptosis of CLL B cells. Free iC3b levels were elevated in CLL patients indicating that this ligand is available in vivowhere it may interact with beta2 integrins on CLL B cells and sustain their viability by preventing activation of the programmed cell death pathway. Leukemia (2000) 14, 34-39.

Expression of CD21 is developmentally regulated during thymic maturation of human T lymphocytes.

CD21, the C3d/CD23/Epstein-Barr virus (EBV), receptor is expressed at low density on cells of the T lineage. Immature thymocytes express CD21 with high density. In the present study, we have analyzed the expression of CD21 during intrathymic maturation of T cells. An intense staining for CD21 was observed at the double-negative stage and at the stage of early acquisition of CD4. CD21 expression was decreased at the double-positive and single-positive stages, to then reach levels similar to those of peripheral blood T cells. Staining of thymus sections showed a bright fluorescent signal on thymocytes entering the thymus in the cortical region. Taking advantage of the immature phenotype of cells expressing high amounts of CD21 (CD21(++)), we depleted thymocyte suspensions in CD3(+) and CD8(+) cells to study the properties of CD21 on this cell subset. Triggering of CD21 with its ligands iC3b, CD23 and anti-CD21 mAb did not alter the proliferative response of thymocytes to IL-7, and did not induce the differentiation of early cells into CD4(+)CD8(+) thymocytes. Immunoprecipitation did not reveal any molecule associated with CD21 that could play a signaling role in thymocytes. Finally, EBV induced a down-regulation of CD21 and an up-regulation of CD1 in CD21(++) thymocytes. Taken together, our observations demonstrate a regulated expression of CD21 on human thymocytes and suggest that the CD21(++) subset may be a target for EBV. We further suggest that CD21 on early thymocytes acts as a ligand for CD23-expressing cells in the thymus.

The Expression of Adhesion Molecules, including CD11a and CD11b, on the Human T Lymphocyte Surface Membrane in Patients with Bronchial Asthma.

The beta-2 integrin consists of lymphocyte function-antigen-1 (LFA-1, CD11a/CD18) which binds to the major ligand intracellular adhesion molecules 1 and 2 (ICAM-1 and ICAM-2), Mac-1 (CD11b/CD18, ligand iC3b) and p150,95 (CD11c/CD18, ligand iC3b). These molecules function not only as adhesion molecules but as signal transduction agents. To investigate the expression of an integrin family in asthma, lymphocyte subsets were assessed using bronchoalveolar lavage fluid (BALF) cells obtained from asthma patients. The relationship between the integrins and lymphocyte subpopulations and their activation state in the BALF was analyzed using two-color flow cytometry in 15 subjects with asthma and in 13 control subjects with chronic bronchitis. The absolute number of CD11a+CD4+ lymphocytes was significantly greater in the asthmatics than in the nonsmoking subjects with chronic bronchitis. In the asthmatics, the expression of the CD11a+CD4+ lymphocytes was significantly higher than the expression of CD11a+CD8+ lymphocytes. The CD2+ lymphocytes had a significant positive regression with the CD11a+CD4+ lymphocytes in the asthmatics. These findings indicate that the CD11a+ antigen is mainly located on the CD4+ lymphocytes in asthmatics, and that the subset of CD11a lymphocytes with the CD4+ antigen increases as the absolute number of T lymphocytes increases. Analysis of BALF revealed the representation of integrins for the ongoing bronchial transfer of information, and may provide an approach to obtain evidence about the inflammatory processes on the surface of the bronchioles in asthma.

The A-domain of beta 2 integrin CR3 (CD11b/CD18) is a receptor for the hookworm-derived neutrophil adhesion inhibitor NIF.

The A-domain is a approximately 200-amino acid peptide present within structurally diverse proadhesive proteins including seven integrins. A recombinant form of the A-domain of beta 2 integrins CR3 and LFA-1 has been recently shown to bind divalent cations and to contain binding sites for protein ligands that play essential roles in leukocyte trafficking to inflammatory sites, phagocytosis and target cell killing. In this report we demonstrate that the neutrophil adhesion inhibitor, NIF produced by the hookworm Ancyclostoma caninium is a selective CD11b A-domain binding protein. NIF bound directly, specifically and with high affinity (Kd of approximately 1 nM) to recombinant CD11b A-domain (r11bA). The binding reaction was characterized by rapid association and very slow dissociation, and was blocked by an anti-r11bA monoclonal antibody. No binding was observed to rCD11aA. The NIF-r11bA interaction required divalent cations, and was absent when the mutant r11bA D140GS/AGA (that lacks divalent cation binding capacity) was used. The NIF binding site in r11bA was mapped to four short peptides, one of which being an iC3b binding site. The interaction of NIF with CR3 in intact cells followed similar binding kinetics to those with r11bA, and occurred with similar affinity in resting and activated human neutrophils, suggesting that the NIF epitope is activation independent. Binding of NIF to CR3 blocked its ability to bind to its ligands iC3b, fibrinogen, and CD54, and inhibited the ability of human neutrophils to ingest serum opsonized particles. NIF thus represents the first example of a disintegrin that targets the integrin A-domain, and is likely to be used by the hookworm to evade the host's inflammatory response. The unique structure of NIF, which lacks a disintegrin motif, emphasizes basic structural differences in antagonists targeting A+ and A- integrins, that should be valuable in drug design efforts aimed at generating novel therapeutics. Identification of the region in NIF mediating A-domain binding should also be useful in this regard, and may, as in the case of disintegrins, unravel a new structural motif with cellular counterparts mediating important physiologic functions.

CR3 (Mac-1, alpha M beta 2, CD11b/CD18) and Fc gamma RIII cooperate in generation of a neutrophil respiratory burst: requirement for Fc gamma RIII and tyrosine phosphorylation.

Cooperation among plasma membrane receptors in activating signal transduction cascades is not well understood. For almost 20 years, it has been clear that when a particulate foreign body is opsonized with complement as well as IgG, the efficiency of IgG effector functions is markedly enhanced. However, the molecular mechanisms involved in cooperation between IgG Fc receptors and complement receptors have not been elucidated. In this work, we show that when human neutrophils (PMN) are plated on a surface coated with both anti-CR3 and anti-Fc gamma RIII antibodies, the respiratory burst which occurs is equivalent to that stimulated by anti-Fc gamma RII. The CR3 ligand iC3b is as effective as anti-CR3 for cooperating with anti-Fc gamma RIII in generation of a respiratory burst. The synergy between CR3 and Fc gamma RIII for activating the NADPH oxidase is abolished by Fab of anti-Fc gamma RII. Nonetheless, the observed synergy is not an artifact of unintended Fc gamma RII ligation, since (a) only this combination of antibodies works to generate H2O2; (b) coating plates with either of the antibodies alone cannot activate the respiratory burst at any dose; (c) LAD (CR3 deficient) cells, which are perfectly competent to mount a respiratory burst when Fc gamma RII is engaged, are incapable of activating the respiratory burst when adherent to wells coated with anti-Fc gamma RIII and anti-CR3; (d) direct engagement of Fc gamma RII activates the respiratory burst by a pathway pharmacologically distinguishable from the synergistic respiratory burst. Fc gamma RIII/CR3 synergy is abolished by cytochalasin B and herbimicin, suggesting that both the actin cytoskeleton and tyrosine phosphorylation are necessary for activation of the synergistic respiratory burst. Further analysis shows that CR3 and Fc gamma RIII have distinct roles in activation of this Fc gamma RII-dependent assembly of the NADPH oxidase. Ligation of CR3 is sufficient to lead to Fc gamma RII association with the actin cytoskeleton on the adherent PMN surface. Coligation of Fc gamma RIII is required for tyrosine phosphorylation of Fc gamma RII. These data are consistent with a model in which phosphorylation of Fc gamma RII or a closely associated substrate initiates activation of a signal transduction pathway leading to oxidase assembly. These are the first data to demonstrate a molecular mechanism for synergy between IgG Fc and complement receptors in activation of phagocyte effector functions.

Molecular mimicry in Candida albicans. Role of an integrin analogue in adhesion of the yeast to human endothelium.

Hematogenous infection with the yeast Candida albicans now occurs with increasing frequency in the neonate, the immunocompromised patient, and the hyperglycemic or hyperalimented host. Yeast-phase C. albicans expresses a protein that is antigenically and structurally related to CD11b/CD18, a member of the beta 2 integrins and a well-characterized adhesin for mammalian neutrophils. Both the neutrophil protein and its analogue in C. albicans have an identical affinity for the C3 ligand iC3b, and both proteins are significantly increased in expression at 37 degrees C. Given these several similarities, we therefore studied the role of the integrin analogue on C. albicans in the adhesion of the yeast to human umbilical vein endothelium (HUVE). After growth of C. albicans in 20 mM D-glucose, as opposed to 20 mM L-glutamate, flow cytometric analysis with monoclonal antibodies recognizing the alpha-subunit of CD11b/CD18 demonstrated a 25.0% increase in mean channel fluorescence (range 18.4-31.8%), as well as an increased percentage of yeasts fluorescing (P less than 0.02). This increased intensity of fluorescence, which corresponds to increased expression of the integrin analogue, also correlated with a significant increase of 30-80% in adhesion of glucose-grown C. albicans to HUVE (P less than 0.02). Blockade of the integrin analogue on C. albicans by monoclonal antibodies recognizing adhesive epitopes on neutrophil CD11b/CD18 inhibited glucose-enhanced adhesion of C. albicans to HUVE. Incubation of glucose-grown C. albicans with saturating concentrations of purified human iC3b, the ligand for CD11b/CD18, reduced adhesion of the yeast to HUVE by 49.7%, whereas BSA in equimolar concentration had no effect (P less than 0.001). These results identify a glucose-responsive integrin analogue on C. albicans as one of possibly several cellular structures that mediate adhesion of the yeast to human endothelium.

Isolation and characterization of cell surface mutants of Candida albicans

Mutant strains of Candida albicans were obtained by selecting for cells that escaped agglutination by a polyclonal antiserum raised against standard C. albicans serotype A isolate B311. Mutants were obtained from strains B311 and B792 and from four strains isolated from patients with acquired immunodeficiency syndrome. All 15 tested mutants retained characteristic sugar assimilation patterns. All but one of the mutants retained the ability to form germ tubes and chlamydospores. Two mutants from an acquired immunodeficiency syndrome-derived isolate were deficient in binding complement ligands iC3b and C3d, whereas another mutant was deficient in binding ligand iC3b but not C3d. The hyphae of these three mutants lacked antigens when examined by Western immunoblotting with monoclonal antibody Ca-A, which detects several glycoproteins, including C3d-binding proteins. One of the complement-binding-deficient mutants was tested for its ability to colonize the gastrointestinal tract of rabbits but did not differ from the wild-type parent in site or degree of colonization. The proton magnetic resonance spectra of bulk mannan carbohydrate extracted from tested mutants showed the loss of a signal characteristic of the mannosyl alpha-PO4 linkage; each mutant also had a distinct pattern of other changes.