Robert B. Sim

Distinct Pathways of Mannan-Binding Lectin (MBL)- and C1-Complex Autoactivation Revealed by Reconstitution of MBL with Recombinant MBL-Associated Serine Protease-2

Mannan-binding lectin (MBL) plays a pivotal role in innate immunity by activating complement after binding carbohydrate moieties on pathogenic bacteria and viruses. Structural similarities shared by MBL and C1 complexes and by the MBL- and C1q-associated serine proteases, MBL-associated serine protease (MASP)-1 and MASP-2, and C1r and C1s, respectively, have led to the expectation that the pathways of complement activation by MBL and C1 complexes are likely to be very similar. We have expressed rMASP-2 and show that, whereas C1 complex autoactivation proceeds via a two-step mechanism requiring proteolytic activation of both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. The results suggest that the catalytic activities of MASP-2 split between the two proteases of the C1 complex during the course of vertebrate complement evolution.

Human erythrocytes bind and inactivate type 5 adenovirus by presenting Coxsackie virus-adenovirus receptor and complement receptor 1

Type 5 adenovirus (Ad5) is a human pathogen that has been widely developed for therapeutic uses, with only limited success to date. We report here the novel finding that human erythrocytes present Coxsackie virus-adenovirus receptor (CAR) providing an Ad5 sequestration mechanism that protects against systemic infection. Interestingly, erythrocytes from neither mice nor rhesus macaques present CAR. Excess Ad5 fiber protein or anti-CAR antibody inhibits the binding of Ad5 to human erythrocytes and cryo-electron microscopy shows attachment via the fiber protein of Ad5, leading to close juxtaposition with the erythrocyte membrane. Human, but not murine, erythrocytes also present complement receptor (CR1), which binds Ad5 in the presence of antibodies and complement. Transplantation of human erythrocytes into nonobese diabetic/severe combined immunodeficiency mice extends blood circulation of intravenous Ad5 but decreases its extravasation into human xenograft tumors. Ad5 also shows extended circulation in transgenic mice presenting CAR on their erythrocytes, although it clears rapidly in transgenic mice presenting erythrocyte CR1. Hepatic infection is inhibited in both transgenic models. Erythrocytes may therefore restrict Ad5 infection (natural and therapeutic) in humans, independent of antibody status, presenting a formidable challenge to Ad5 therapeutics. “Stealthing” of Ad5 using hydrophilic polymers may enable circumvention of these natural virus traps.

Carbohydrate-independent recognition of collagens by the macrophage mannose receptor

Mannose receptor (MR) is the best characterised member of a family of four endocytic molecules that share a common domain structure; a cysteine‐rich (CR) domain, a fibronectin‐type II (FNII) domain and tandemly arranged C‐type lectin‐like domains (CTLD, eight in the case of MR). Two distinct lectin activities have been described for MR. The CR domain recognises sulphated carbohydrates while the CTLD mediate binding to mannose, fucose or N‐acetylglucosamine. FNII domains are known to be important for collagen binding and this has been studied in the context of two members of the MR family, Endo180 and the phospholipase A2 receptor. Here, we have investigated whether the broad and effective lectin activity mediated by the CR domain and CTLD of MR is favoured to the detriment of FNII‐mediated interaction(s). We show that MR is able to bind and internalise collagen in a carbohydrate‐independent manner and that MR deficient macrophages have a marked defect in collagen IV and gelatin internalisation. These data have major implications at the molecular level as there are now three distinct ligand‐binding sites described for MR. Furthermore our findings extend the range of endogenous ligands recognised by MR, a molecule firmly placed at the interface between homeostasis and immunity.

The Glycosylation of Human Serum IgD and IgE and the Accessibility of Identified Oligomannose Structures for Interaction with Mannan-Binding Lectin

Analysis of the glycosylation of human serum IgD and IgE indicated that oligomannose structures are present on both Igs. The relative proportion of the oligomannose glycans is consistent with the occupation of one N-linked site on each heavy chain. We evaluated the accessibility of the oligomannose glycans on serum IgD and IgE to mannan-binding lectin (MBL). MBL is a member of the collectin family of proteins, which binds to oligomannose sugars. It has already been established that MBL binds to other members of the Ig family, such as agalactosylated glycoforms of IgG and polymeric IgA. Despite the presence of potential ligands, MBL does not bind to immobilized IgD and IgE. Molecular modeling of glycosylated human IgD Fc suggests that the oligomannose glycans located at Asn354 are inaccessible because the complex glycans at Asn445 block access to the site. On IgE, the additional CH2 hinge domain blocks access to the oligomannose glycans at Asn394 on one H chain by adopting an asymmetrically bent conformation. IgE contains 8.3% Man5GlcNAc2 glycans, which are the trimmed products of the Glc3Man9GlcNAc2 oligomannose precursor. The presence of these structures suggests that the CH2 domain flips between two bent quaternary conformations so that the oligomannose glycans on each chain become accessible for limited trimming to Man5GlcNAc2 during glycan biosynthesis. This is the first study of the glycosylation of human serum IgD and IgE from nonmyeloma proteins.

Recognition of Candida albicans by mannan-binding lectin in vitro and in vivo

Mannan-binding lectin (MBL) is a component of the innate immune system. The goal of the present study was to evaluate binding of MBL to Candida albicans in vitro and in vivo and to assess the impact of MBL treatment on host resistance. The results showed a variable and often discontinuous pattern of binding to individual yeast cells. MBL bound to cells grown at 37 degrees C but not to cells grown at 23 degrees C. The putative MBL ligand was constitutively present on yeast cells grown at 23 degrees C, but the ligand was masked on such cells, such that MBL could not bind. C. albicans yeasts and hyphae in infected tissue bound MBL. Finally, parenteral administration of MBL increased resistance of mice to hematogenously disseminated candidiasis. These results suggest that MBL is an important component of innate resistance to candidiasis and that MBL therapy may be a means to prevent disseminated candidiasis in high-risk patients.

Scrapie Pathogenesis: The Role of Complement C1q in Scrapie Agent Uptake by Conventional Dendritic Cells

Mice lacking complement components show delayed development of prion disease following peripheral inoculation. The delay could relate to reduced scrapie prion protein (PrPSc) accumulation on follicular dendritic cells (DCs). However conventional DCs (cDCs) play a crucial role in the early pathogenesis of prion diseases and complement deficiency could result in decreased PrPSc uptake by cDCs in the periphery. To explore this possibility, we cultured murine splenic or gut-associated lymph node cDCs with scrapie-infected whole brain homogenate in the presence or absence of complement. Uptake decreased significantly if the serum in the cultures was heat-inactivated. Because heat inactivation primarily denatures C1q, we used serum from C1q−/− mice and showed that PrPSc uptake was markedly decreased. PrPSc internalization was saturable and temperature-dependent, suggesting receptor-mediated uptake. Furthermore, uptake characteristics differed from fluid-phase endocytosis. Immunofluorescence showed colocalization of C1q and PrPSc, suggesting interaction between these molecules. We evaluated the expression of several complement receptors on cDCs and confirmed that cDCs that take up PrPSc express one of the C1q receptors, calreticulin. Our results show that C1q participates in PrPSc uptake by cDCs, revealing a critical role for cDCs in initial prion capture, an event that takes place before the PrPSc accumulation within the follicular DC network.

Characterization of radioiodinated lung surfactant protein A (SP-A) and the effects of oxidation on SP-A quaternary structure and activity

Lung surfactant protein A (SP-A) is the most abundant surfactant-associated protein present in the lung and respiratory tract. SP-A binds to several pathogens via its C-type lectin domains, and may act as an opsonin, mediating adhesion to cells via the collectin receptor. Binding studies using SP-A are made difficult by its apparent instability following radioiodination. This study investigated the effect of oxidation (via radioiodination and exposure to H2O2) on the structural and functional characteristics of SP-A. Radioiodinated SP-A, stored at 4 degrees C, retained carbohydrate binding activity after labeling. After 10 days storage, the radioiodinated SP-A was indistinguishable on SDS-PAGE from freshly radioiodinated SP-A, but sedimentation coefficient and Stokes radius values changed dramatically, indicating SP-A depolymerization. Such a quaternary structural breakdown, with a concomitant reduction in carbohydrate binding activity, is likely to be due to oxidative cleavage of disulfide bonds. Comparable results were observed upon radioiodination of the structurally similar molecule C1q. Consequently, the effect of prolonged incubation with H2O2 upon SP-A was investigated, with similar results. Thus, exposure to oxidizing agents leads to breakdown of the hexameric quaternary structure of SP-A, often to native polypeptides, with an attendant loss of binding activity. Such an effect may have consequences for the physiological role of SP-A in the lung.

Molecular Aspects of Innate and Adaptive Immunity

Molecular aspects of innate and adaptive immunity , Molecular aspects of innate and adaptive immunity , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

Molecular Biology of the Human Complement Class III Products of the MHC: Factor I and Its Cofactors

Deletion analysis of the 5′ flanking region of the factor B gene indicates the presence of a region with enhancer-like activity in addition to the promoter element and indicates that both elements are important in the tissue-specific expression of factor B. DNA sequencing of a number of C4 allotypes has allowed definition of the structural basis of the polymorphism found in C4A and C4B types in terms of gene size, reactivity, and antigenicity. It has been established that the regulatory proteins C4bp, CR1, and H as well as the enzymes C2, B, and C1r are structurally related since they contain two or more internal repeating units of 60 amino acids. Thus these complement proteins appear to be members of a new superfamily since this structural feature is widespread, being found in a number of noncomplement proteins.