Maria Rosa Sarrias

Complement and innate immunity

Wen-Chao Song , Maria Rosa Sarrias , John D. Lambris b,) a Center for Experimental Therapeutics and Department of Pharmacology, UniOersity of PennsylOania School of Medicine, 1351 BRBIIr III, 421 Curie BlOd., Philadelphia, PA 19104, USA b Protein Chemistry Laboratory, Department of Pathology and Laboratory Medicine, UniOersity of PennsylOania School of Medicine, 401 Stellar Chance Laboratories, Philadelphia, PA 19104, USA

Relevance of CD6-mediated interactions in T cell activation and proliferation.

CD6 is a cell surface receptor expressed on immature thymocytes and mature T and B1a lymphocytes. The ultimate function of CD6 has not been deciphered yet, but much evidence supports a role for CD6 in T cell activation and differentiation. In this study, we show that a fraction of CD6 molecules physically associates with the TCR/CD3 complex by coimmunoprecipitation, cocapping, and fluorescence resonance energy transfer experiments. Image analysis of Ag-specific T-APC conjugates demonstrated that CD6 and its ligand, activated leukocyte cell adhesion molecule (CD166), colocalize with TCR/CD3 at the center of the immunological synapse, the so-called central supramolecular activation cluster. The addition of a soluble rCD6 form significantly reduced the number of mature Ag-specific T-APC conjugates, indicating that CD6 mediates early cell-cell interactions needed for immunological synapse maturation to proceed. This was in agreement with the dose-dependent inhibition of CD3-mediated T cell proliferation induced by soluble rCD6. Taken together, our data illustrate the important role played by the intra- and intercellular molecular interactions mediated by CD6 during T cell activation and proliferation processes.

Cloning and structure of three rainbow trout C3 molecules: a plausible explanation for their functional diversity ☆

We have previously identified and characterized three distinct trout C3 proteins (C3-1, C3-3 and C3-4) that differ in their electrophoretic mobility, glycosylation patterns, reactivity with monospecific C3 antibodies, partial amino acid sequence and binding to various complement activators. To study the structural elements that determine the observed functional differences, we have cloned and sequenced the three C3 isoforms. Comparison of the deduced amino acid sequences showed that the sequence identity/similarity of C3-3 to C3-4 is 76/81%, whereas those of C3-3 and C3-4 to C3-1 are 55/67% and 54/67%, respectively. It is interesting that the beta-chain of C3-4 contains two insertions of 65 (residues 504-569) and 23 amino acids (residues 123-146), while the beta-chain of C3-1 contains a 14-amino acid insertion (residues 143-157). The C3 convertase cleavage site (Arg-Ser) is conserved in the three trout isoforms; however, the factor I cleavage sites are Arg-Ala (for C3-1 and C3-4) and Arg-Thr (C3-3) instead of Arg-Ser at position 1281 of human C3, and Arg-Thr (C3-1, C3-3) instead of Arg-Ser for C3-4 at position 1298 of human C3. Of special interest is the absence of the His(1126) and Glu(1128) (human C3 numbering) from C3-4 and of Glu(1128) from C3-3. These residues are thought to play an important role in determining the binding specificity of the thioester-containing proteins. Accordingly, we postulate that the distinct binding reactions of the trout C3 isoforms with various complement activators could be due at least in part to the observed changes in the His and Glu residues.

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.

The three HveA receptor ligands, gD, LT-α and LIGHT bind to distinct sites on HveA

Abstract The herpes virus entry mediator A (HveA), a member of the tumor necrosis factor receptor (TNFR) superfamily, interacts with three different protein ligands; lymphotoxin-α (LT-α) and LIGHT (LIGHT stands for l ymphotoxin homolog, which exhibits i nducible expression and competes with HSV g lycoprotein D for H veA and is expressed on T -lymphocytes) from the host and the herpes simplex virus (HSV) surface glycoprotein gD. It has been reported that the gD binding site on HveA is located within the receptors two N-terminal CRP domains, and that gD and LIGHT compete for their binding to HveA. However, whether these ligands interact with the same or different sites on the receptor is unclear. We analyzed and compared the sites of interaction between HveA and its TNF ligands, by using two recombinant forms of the receptor, comprising the full-receptor ectodomain (HveA (200t)) and its two first CRP domains (HveA (120t)), as well as several monoclonal antibodies recognizing HveA. Two HveA peptide ligands (BP-1 and BP-2) that differentially inhibit binding of soluble gD and LT-α to the receptor were also used to demonstrate that gD, LIGHT and LT-α bind to distinct sites on the receptor. Our results suggest that binding of a ligand to HveA may alter the conformation of this receptor, thereby affecting its interaction with its other ligands.

Studies of Structure-Activity Relations of Complement Inhibitor Compstatin.

Compstatin, a 13-mer cyclic peptide, is a novel and promising inhibitor of the activation of the complement system. In our search for a more active analog and better understanding of structure-functions relations, we designed a phage-displayed random peptide library based on previous knowledge of structure activity relations, in which seven amino acids deemed necessary for structure and activity were kept fixed while the remaining six were optimized. Screening of this library against C3 identified four binding clones. Synthetic peptides corresponding to these clones revealed one analog, called acetylated Ile1Leu/His9Trp/Thr13Gly triple replacement analog of compstatin corresponding to clone 640 (Ac-I1L/H9W/T13G), which was more active than compstatin. This newly identified peptide had 4-fold higher activity when compared with the originally isolated form of compstatin and 1.6-fold higher activity when compared with acetylated compstatin (Ac-compstatin). The structures of Ac-I1L/H9W/T13G and Ac-compstatin were studied by nuclear magnetic resonance, compared with the structure of compstatin, and found to be very similar. The binding of Ac-I1L/H9W/T13G and the equally active acetylated analog with His9Ala replacement (Ac-H9A) to C3 was evaluated by surface plasmon resonance, which suggested similarity in their binding mechanism but difference when compared with Ac-compstatin. Compensatory effects of flexibility outside the β-turn and tryptophan ring stacking may be responsible for the measured activity increase in Ac-I1L/H9W/T13G and acetylated analog with His9Ala replacement and the variability in binding mechanism compared with Ac-compstatin. These data demonstrate that tryptophan is a key amino acid for activity. Finally, the significance of the N-terminal acetylation was examined and it was found that the hydrophobic cluster at the linked termini of compstatin is essential for binding to C3 and for activity.

Expression of Interleukin-8 Receptors (CXCR1 and CXCR2) in Premenopausal Women with Recurrent Urinary Tract Infections

ABSTRACT The migration of neutrophils through infected tissues is mediated by the CXC chemokines and its receptors (CXCR1 and CXCR2). It has been proposed that a CXCR1 deficiency could confer susceptibility to acute pyelonephritis in children. The objective of the study is to assess the surface expression of CXCR1 and CXCR2 and the existence of polymorphisms in the CXCR1 gene in premenopausal women with recurrent urinary tract infections. The study included 20 premenopausal women with recurrent urinary infections, with normal urinary tracts, and without diseases potentially associated with relapsing urinary infections and 30 controls without previous urinary infections. The levels of CXCR1 and CXCR2 expression on neutrophils were measured and analyzed by flow cytometry by measuring the mean fluorescence intensity (MFI) channel. The promoter and coding regions of the CXCR1 gene were analyzed for the presence of polymorphisms by a sequence-based typing method. Patients with recurrent urinary tract infections exhibited median levels of CXCR1 expression, determined from MFI values, similar to those of the controls. The analysis of CXCR2 showed that patients with recurrent urinary infections had lower median levels of expression, determined from the MFI values, than the controls (P = 0.002, Mann-Whitney U test). No polymorphisms were detected at the promoter or at the exon 1 region of the CXCR1 gene either in the patients or in the controls. Polymorphisms were detected at the exon 2 of CXCR1, but their frequencies did not differ between patients and controls. We have found a low level of CXCR2 expression in patients with recurrent urinary tract infections. These results suggest that a low level of CXCR2 expression may increase the susceptibilities of premenopausal women to urinary tract infections.

Crystal Structure of the Third Extracellular Domain of CD5 Reveals the Fold of a Group B Scavenger Cysteine-rich Receptor Domain

Scavenger receptor cysteine-rich (SRCR) domains are ancient protein modules widely found among cell surface and secreted proteins of the innate and adaptive immune system, where they mediate ligand binding. We have solved the crystal structure at 2.2 Å of resolution of the SRCR CD5 domain III, a human lymphocyte receptor involved in the modulation of antigen specific receptor-mediated T cell activation and differentiation signals. The first structure of a member of a group B SRCR domain reveals the fold of this ancient protein module into a central core formed by two antiparallel β-sheets and one α-helix, illustrating the conserved core at the protein level of genes coding for group A and B members of the SRCR superfamily. The novel SRCR group B structure permits the interpretation of site-directed mutagenesis data on the binding of activated leukocyte cell adhesion molecule (ALCAM/CD166) binding to CD6, a closely related lymphocyte receptor homologue to CD5.

Structure, functions, and evolution of the third complement component and viral molecular mimicry

The third component of the complement system, C3, is a common denominator in the activation of the classical, alternative, and lectin pathways. The ability of C3 molecule to interact with at least 20 different proteins makes it the most versatile component of this system. Since these interactions are important for phagocytic, immunoregulatory, and immune evasion mechanisms, the analysis of its structure and functions has been a subject of intense research. Here we review our current work on the C3-ligand interactions, C3-related viral molecular mimicry, evolution of the complement system, and identification of C3-based complement inhibitors.

Cloning, structure, and function of two rainbow trout Bf molecules: Both classical and alternative pathway activities require the presence of Bf-2☆

The factor B (Bf) and C2 complement genes are closely linked within the MHC class III region and are thought to have arisen by gene duplication from a single gene encoding an ancestral molecule; the animal phyla in which this duplication event took place is unknown. Two teleost fish, (zebrafish and medaka fish) have each been shown to possess only a single molecule that shows an equivalent degree of similarity to mammalian Bf and C2. In contrast, here we present the characterization of two factor B molecules (Bf-1 and Bf-2) in another teleost fish (the rainbow trout) that are about 9% more similar to mammalian factor B than C2, yet play a role in both alternative and classical pathways of complement activation. The full lengths of Bf-1 and Bf-2 cDNAs are 2509 and 2560 bp, respectively, and their deduced amino acid sequences are 75% identical. Both trout Bf genes are mainly expressed in liver and appear to be single-copy genes. The isolated Bf-1 and Bf-2 proteins are able to form the alternative pathway C3 convertase and are cleaved (in the presence of purified trout C3, trout factor D, and Mg2+ EGTA) into Ba- and Bb-like fragments in a manner similar to that seen for mammalian factor B. The most remarkable feature of trout Bf-2 is its ability to restore the hemolytic activity of trout Bf-depleted serum through both the alternative and classical pathways; whether Bf-1 possess similar activity is unclear at present.