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Abstract: The first eight and the last two of 20 complement control protein (CCP) modules within complement factor H (fH) encompass binding sites for C3b and polyanionic carbohydrates. These binding sites cooperate self-surface selectively to prevent C3b amplification, thus minimising complement-mediated damage to host. Intervening fH CCPs, apparently devoid of such recognition sites, are proposed to play a structural role. One suggestion is that the generally small CCPs 10–15, connected by longer-than-average linkers, act as a flexible tether between the two functional ends of fH; another is that the long linkers induce a 180° bend in the middle of fH. To test these hypotheses, we determined the NMR-derived structure of fH12–13 consisting of module 12, shown here to have an archetypal CCP structure, and module 13, which is uniquely short and features a laterally protruding helix-like insertion that contributes to a prominent electropositive patch. The unusually long fH12–13 linker is not flexible. It packs between the two CCPs that are not folded back on each other but form a shallow vee shape; analytical ultracentrifugation and X-ray scattering supported this finding. These two techniques additionally indicate that flanking modules (within fH11–14 and fH10–15) are at least as rigid and tilted relative to neighbours as are CCPs 12 and 13 with respect to one another. Tilts between successive modules are not unidirectional; their principal axes trace a zigzag path. In one of two arrangements for CCPs 10–15 that fit well with scattering data, CCP 14 is folded back onto CCP 13. In conclusion, fH10–15 forms neither a flexible tether nor a smooth bend. Rather, it is compact and has embedded within it a CCP module (CCP 13) that appears to be highly specialised given both its deviant structure and its striking surface charge distribution. A passive, purely structural role for this central portion of fH is unlikely. [Copyright &y& Elsevier]

Human plasma protein complement factor H (FH) is an inhibitor of the spontaneously activated alternative complement pathway. An allotypic variant of FH, 402His, has been associated with age-related macular degeneration, the leading cause of blindness in the elderly. Crystals of FH domains 6–8 (FH678) containing 402His have been grown in the presence of a polyanionic sucrose octasulfate ligand (an analogue of the natural glycosaminoglycan ligands of FH) using both native and selenomethionine-derivatized protein. Native data sets diffracting to 2.3 Å and SeMet data sets of up to 2.8 Å resolution have been collected. An anomalous difference Patterson map reveals self- and cross-peaks from two incorporated Se atoms. The corresponding selenium substructure has been solved. [ABSTRACT FROM AUTHOR]

Abstract: Human CD46 (membrane cofactor protein) has recently been identified to be an attachment receptor for subgroup B adenoviruses (Ads); however, the precise interaction between human CD46 and subgroup B Ads are just beginning to be understood. In this study, to characterize the interaction between human CD46 and subgroup B Ads, varieties of mutant CD46 were tested for their ability to act as a receptor for Ad serotype 35 (Ad35), which belongs to subgroup B. In addition, we determined Ad35 vector-mediated transgene expression and cellular uptake of Ad35 vectors in the presence of a set of anti-CD46 antibodies. Our data demonstrated that the short consensus repeats (SCRs) 1 and 2 in human CD46 are important for interaction with Ad35, whereas the cytoplasmic domain of human CD46 was found not to be required for the function as an Ad35 receptor. Rather, a complete deletion of the cytoplasmic domain of human CD46 increased the transduction efficiencies of Ad35 vectors. This information should help in elucidation of the mechanism of subgroup B Ad infection, as well in the improvement of the subgroup B Ad vectors. [Copyright &y& Elsevier]

The gene encoding the human plasma protein β2‐glycoprotein I or apolipoprotein H was cloned and its structure determined. The gene which consists of eight exons was shown to span 18 kb and was localized to chromosome 17q23‐24. The transcriptional initiation site was assigned to a position 31 bp upstream of the start codon. Several consensus sequence elements relevant for regulation of transcription in liver were seen in the 5′‐upstream region of the gene. Exon 1 contains the 5′‐UTR together with the signal peptide coding sequences. Short consensus repeats (SCRs) 1, 3, 4, and 5 are encoded by single exons each while SCR2 is encoded by two exons. Exon 8 comprises the region encoding the C‐terminal end of β2‐glycoprotein I (from His‐310), the stop codon and the 3′‐UTR. [ABSTRACT FROM AUTHOR]

The high affinity of human plasma Β2-glycoprotein I (Β2GPI), also known as apolipoprotein-H (Aport), for negatively charged phospholipids determines its implication in a variety of physiological pathways, including blood coagulation and the immune response. Β2GPI is considered to be a cofactor for the binding of serum autoantibodies from antiphospholipid syndrome (APS) and correlated with thrombosis, lupus erythematosus and recurrent fetal loss. We solved the Β2GPI structure from a crystal form with 84% solvent and present a model containing all 326 amino acid residues and four glycans. The structure reveals four complement control protein modules and a distinctly folding fifth C-terminal domain arranged like beads on a string to form an elongated J-shaped molecule. Domain V folds into a central Β-spiral of four antiparallel Β-sheets with two small helices and an extended C-terminal loop region. It carries a distinct positive charge and the sequence motif CKNKEKKC close to the hydrophobic loop composed of residues LAFW (313–316), resulting in an excellent counterpart for interactions with negatively charged amphiphilic substances. The Β2GPI structure reveals potential autoantibody-binding sites and supports mutagenesis studies where Trp316 and CKNKEKKC have been found to be essential for the phospholipid-binding capacity of Β2GPI. [ABSTRACT FROM AUTHOR]

Human β2-glycoprotein I is a heavily glycosylated five-domain plasma membrane-adhesion protein, which has been implicated in blood coagulation and clearance of apoptotic bodies from the circulation. It is also the key antigen in the autoimmune disease anti­phospholipid syndrome. The crystal structure of β2-glycoprotein I isolated from human plasma revasls an elongated fish-hook-like arrangement of the globular short consensus repeat domains. Half of the C-terminal fifth domain deviates strongly from the standard fold, as observed in domains one to four. This aberrant half forms a specific phospholipid­binding sits. A large patch of 14 positively charged residues provides electrostatic interactions with anionic phospholipid head groups and an exposed membrane-insertion loop yields specificity for lipid layers. The observed spatial arrangement of the five domains suggests a functional partitioning of protein adhesion and membrane adhesion over the N- and C-terminal domains, respectively, separated by glycosylated bridging domains. Coordinates are in the Protein Data Bank (accession No. 1QUB). [ABSTRACT FROM AUTHOR]

Measles virus is a paramyxovirus which, like other members of the family such as respiratory syncytial virus, is a major cause of morbidity and mortality worldwide. The cell surface receptor for measles virus in humans is CD46, a complement cofactor. We report here the crystal structure at 3.1 Å resolution of the measles virus-binding fragment of CD46. The structure reveals the architecture and spatial arrangement of two glycosylated short consensus repeats with a pronounced interdomain bend and some flexibility at the domain interface. Amino acids involved in measles virus binding define a large, glycan-free surface that extends from the top of the first to the bottom of the second repeat. The extended virus-binding surface of CD46 differs strikingly from those reported for the human virus receptor proteins CD4 and intercellular cell adhesion molecule-1 (ICAM-1), suggesting that the CD46 structure utilizes a novel mode of virus recognition. A highly hydrophobic and protruding loop at the base of the first repeat bears a critical virus-binding residue, thereby defining an important recognition epitope. Molecules that mimic the conformation of this loop potentially could be effective anti-viral agents by preventing binding of measles virus to CD46. [ABSTRACT FROM AUTHOR]

Although the cDNA of human decay accelerating factor (DAF) which restricts complement activation on homologous cell membranes was cloned in 1987, all trials to detect the cDNA of mouse DAF by cross-hybridization were unsuccessful. However, by immunoscreening with a rabbit antiserum against purified mouse DAF, we successfully cloned the cDNA. It contains four typical short consensus repeats (SCR) similar to that In human and guinea pig DAF. The base sequence showed 63.7 and 63.8% identIty to that of human and guinea pig DAF respectively. The deduced amino acid sequence identity to human and guinea pig DAF was 47.2 and 46.5% respectively. Mouse complement receptor related gene Y (Crry)/p65 function is comparable to DAF. SCR3 and SCR4 of mouse DAF showed 50% identity to SCR2 and SCR3 of Crty/p65 respectively. IdentificatIon of the mouse DAF gene should open a new approach for determining the actual in vlvo role of DAF by analyzing autoimmune mice as well as generating DAF gene knockout mice using embryonic stem cells. [ABSTRACT FROM AUTHOR]

Mannose-binding protein (MBP) plays an important role in host defense by recognizing sugar residues on certain pathogens and activating the complement cascade. Recently, we described a new protease, designated MBP-assoclated serine protease (MASP) which is required for complement activation by MBP. We have cloned the cDNA that encodes this protease and found that the deduced amino acid sequence contains an epidermal growth factor-like domain, two short consensus repeats and a serine protease domain. The overall structure of MASP is similar to serine proteases of the first complement component complex, C1r–C1s. Unlike C1r–C1s, however, MASP has a hlstidlne loop structure common to many serine proteases such as trypsin and chymotrypsin. The MASP gene was mapped on the long arm of chromosome 3 which is different from C1r–C1s as well as from trypsin and chymotrypsin. These findings suggest that MASP may have emerged prior to C1r–C1s from a common ancestor. This implies that MBP–MASP, a complex of lectin and serine protease, presumably evolved prior to adaptive immune recognition involving antibody and the classical complement pathway. [ABSTRACT FROM PUBLISHER]

Murine complement receptor type 2 (MCR2) is homologous to human CR2, whereas murine CR1 (MCR1) is structurally and evolutionary different from human CR1. Since ligand specificities of MCR1 and MCR2 are not completely clarified, we analyzed their functional characteristics to correlate them with structural information obtained in molecular biological studies. MCR1 and MCR2 purified from spleen cells were incubated with thlol-Sepharose bearing murine C3b, IC3b, or C3d in the presence or absence of various anti-MCR1 or-MCR1/MCR2 mAbs. Bound and free MCR1 and MCR2 were quantitated by Western biotting or two-site immunoradiometric assay. MCR2 bound to C3d and IC3b similarly efficiently, and 5-fold less efficiently to C3b. These bindings were completely inhibited by MCR1/MCR2-crossreactive antibodies 7G6 and 4E3. MCR1 bound to C3b efficiently and this was partially inhibited by MCR1-monospecific antibody 8C12, but not by 7G6 and 4E3. Combinations of 8C12 and 7G6 or 4E3 completely inhibited MCR1 binding to C3b. Therefore, two binding sites, one unique to MCR1 and the other shared with MCR2, are involved in MCR1 binding to C3b. MCR1 bound also to C3d and this was completely inhibited by 7G6 and 4E3. The binding of this solubilized MCR1 to C3d was as efficient as that of MCR2 to C3d. It seems, therefore, that the site shared by MCR1 and MCR2 that is recognized by both 7G6 and 4E3 binds to C3d and less efficiently to C3b. These results clarify the ligand specificities of MCR1 and MCR2. [ABSTRACT FROM PUBLISHER]

The enhancing effects of the complement system for humoral immunity have primarily focused upon the recognition of complement-bound foreign antigens by a co-receptor complex of the antigen-specific B cell receptor (BCR) and complement receptor 2 (Cr2). In vivo experiments using Cr2 gene deficient mice (which lack the expression of both the Cr1 and Cr2 proteins) do demonstrate depressed humoral responses to immunization but cannot be used to define specific contributions of the singular Cr1 or Cr2 proteins on B cell functions. To study the effect of a Cr2 deficiency in a Cr1 sufficient environment we created a mouse line in which the alternative splice site required for the expression of the Cr2 isoform was removed. This mouse line, Cr2KO, still expressed Cr1 on B cells but was deficient for the full length Cr2 protein. Surprisingly a new alternative splice within the Cr2 gene created a truncated product that encoded a novel protein termed iCr2 that was expressed on the surface of the cells. The Cr2KO mouse thus provides a new model system for the analysis of Cr1 and Cr2 functions in the immune response of the mouse., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The screening of all atypical haemolytic uraemic syndrome (aHUS) patients for factor H autoantibodies is best practice. However, there is no consensus assay for the reporting of factor H autoantibody titres. In this study, three European complement laboratories with expertise in the field of autoantibody testing address this by systematically evaluating several ELISA methods used for the detection of factor H autoantibodies. All methods tested adequately detect high titre samples. However, this study recommends the Paris method for the detection and reporting of factor H autoantibodies to be used when setting up a factor H autoantibody screen. The importance of individual sample background subtraction in these ELISA tests was established. The use of a relative or arbitrary unit index with a common positive and negative serum allowed for consistent comparison of findings from different test centres. Therefore, it is recommended that a standard arbitrary unit scale based on a titration curve from a common positive anti-serum be adopted to allow future establishment of the relative importance of particular titres of factor H autoantibodies in aHUS. Systematic assay for the presence of factor H autoantibodies in patients using the Paris method will provide the longitudinal analysis needed to fully establish the importance of factor H autoantibodies in disease. This will feed into additional research to clarify whether additional factors have a bearing on the phenotype/outcome of autoimmune aHUS., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

A serine protease, motopsin (prss12), plays a significant role in cognitive function and the development of the brain, since the loss of motopsin function causes severe mental retardation in humans and enhances social behavior in mice. Motopsin is activity-dependently secreted from neuronal cells, is captured around the synaptic cleft, and cleaves a proteoglycan, agrin. The multi-domain structure of motopsin, consisting of a signal peptide, a proline-rich domain, a kringle domain, three scavenger receptor cysteine-rich domains, and a protease domain at the C-terminal, suggests the interaction with other molecules through these domains. To identify a protein interacting with motopsin, we performed yeast two-hybrid screening and found that seizure-related gene 6 (sez-6), a transmembrane protein on the plasma membrane of neuronal cells, bound to the proline-rich/kringle domain of motopsin. Pull-down and immunoprecipitation analyses indicated the interaction between these proteins. Immunocytochemical and immunohistochemical analyses suggested the co-localization of motopsin and sez-6 at neuronal cells in the developmental mouse brain and at motor neurons in the anterior horn of human spinal cords. Transient expression of motopsin in neuro2a cells increased the number and length of neurites as well as the level of neurite branching. Interestingly, co-expression of sez-6 with motopsin restored the effect of motopsin at the basal level, while sez-6 expression alone showed no effects on cell morphology. Our results suggest that the interaction of motopsin and sez-6 modulates the neuronal cell morphology., (Copyright © 2013 Elsevier Inc. All rights reserved.)