The association between complement component 2/complement factor B polymorphisms and age-related macular degeneration: A HuGE review and meta-analysis

The authors performed a systematic review of the association of complement component 2(C2)/complement factor B (CFB) gene polymorphisms with age-related macular degeneration (AMD). In total, data from 19 studies published between 2006 and 2011 were pooled for 4 polymorphisms: rs9332739 and rs547154 in the C2 gene and rs4151667 and rs641153 in the CFB gene. Data extraction and assessments for risk of bias were independently performed by 2 reviewers. Allele frequencies and allele and genotypic effects were pooled. Heterogeneity and publication bias were explored. Pooled minor allele frequencies for all 4 SNPs were between 4.7% and 9.6% for all polymorphisms, except for an Indian population in which the C allele at rs9332739 was the major allele. For the C2 polymorphisms, the minor C allele at rs9332739 and the minor T allele at rs547154 carried estimated relative risks (odds ratios) of 0.55 (95% confidence interval (CI): 0.46, 0.65) and 0.47 (95% CI: 0.39, 0.57), respectively. For the CFB polymorphisms, the minor A alleles at rs4151667 and rs614153 carried estimated risks of 0.54 (95% CI: 0.45, 0.64) and 0.41 (95% CI: 0.34, 0.51), respectively. These allele effects contributed to an absolute lowering of the risk of all AMD in Caucasian populations by 2.0%-6.0%. This meta-analysis provides a robust estimate of the protective association of C2/CFB with AMD.

Expression of complement components and regulators by different subtypes of bone marrow-derived macrophages

Under inflammatory conditions, macrophages can differentiate into different functional subtypes. We show that bone marrow-derived macrophages constitutively express different levels of various complement-related genes. The relative expression levels are C1qb > Crry > CFH > C3 > C1r > CFB > DAF1 > CD59a > C2 > C1INH > C1s > C4. Upon activation, the expression of C1r, C1s, C3, C2, CFB, and C1INH was up-regulated, and CFH, CD59a, and DAF1, down-regulated in M1 (induced by interferon-? + lipopolysaccharides (LPS)) and M2b (induced by immune complex + LPS) macrophages. The expression of C4 and CFH was slightly up-regulated in interleukin (IL)-10-induced M2c macrophages. Complement gene expression in IL-4-induced M2a macrophages was weakly down-regulated as compared to resting M0 macrophages. Higher levels of C3, C1INH, and CFB but lower levels of CFH expression in M1 and M2b macrophage suggests that they may be involved in the alternative pathway of complement activation during inflammation.

The WaaL O-antigen lipopolysaccharide ligase has features in common with metal ion-independent inverting glycosyltransferases(*)

WaaL is a membrane enzyme that catalyzes a key step in lipopolysaccharide (LPS) synthesis: the glycosidic bonding of a sugar at the proximal end of the undecaprenyl-diphosphate (Und-PP) O-antigen with a terminal sugar of the lipid A-core oligosaccharide (OS). Utilizing an in vitro assay, we demonstrate here that ligation with purified Escherichia coli WaaL occurs without adenosine-5'-triphosphate (ATP) and magnesium ions. Furthermore, E. coli and Pseudomonas aeruginosa WaaL proteins cannot catalyze ATP hydrolysis in vitro. We also show that a lysine substitution of the arginine (Arg)-215 residue renders an active protein, whereas WaaL mutants with alanine replacements in the periplasmic-exposed residues Arg-215, Arg-288 and histidine (His)-338 and also the membrane-embedded aspartic acid-389 are nonfunctional. An in silico approach, combining predicted topological information with the analysis of sequence conservation, confirms the importance of a positive charge at the small periplasmic loop of WaaL, since an Arg corresponding to Arg-215 was found at a similar position in all the WaaL homologs. Also, a universally conserved H[NSQ]X(9)GXX[GTY] motif spanning the C-terminal end of the predicted large periplasmic loop and the membrane boundary of the transmembrane helix was identified. The His residue in this motif corresponds to His-338. A survey of LPS structures in which the linkage between O-antigen and lipid A-core OS was elucidated reveals that it is always in the beta-configuration, whereas the sugars bound to Und-PP are in the alpha-configuration. Together, our biochemical and in silico data argue that WaaL proteins use a common reaction mechanism and share features of metal ion-independent inverting glycosyltransferases.

Common Themes in Glycoconjugate Assembly Using the Biogenesis of O-Antigen Lipopolysaccharide as a Model System

The biosynthesis of glycoconjugates is remarkably conserved in all types of cells since the biochemical reactions involved exhibit similar characteristics, which can be summarized as follows: (a) the saccharide moiety is formed as a lipid-linked, membrane-associated glycan; (b) the lipid component in most cases is a polyisoprenoid phosphate; (c) the assembly of the lipid-linked saccharide intermediate depends on reactions taking place at both sides of the cell membrane, which requires the obligatory transmembrane movement of amphipathic molecules across the lipid bilayer. These general characteristics are present in the biosynthesis of the O-antigen component of the bacterial lipopolysaccharide, which serves as a model system to investigate the molecular and mechanistic basis of glycoconjugate synthesis, as summarized in this mini-review.

Structure-Guided Investigation of Lipopolysaccharide O-Antigen Chain Length Regulators Reveals Regions Critical for Modal Length Control

The O-antigen component of the lipopolysaccharide (LPS) represents a population of polysaccharide molecules with nonrandom (modal) chain length distribution. The number of the repeat O units in each individual O-antigen polymer depends on the Wzz chain length regulator, an inner membrane protein belonging to the polysaccharide copolymerase (PCP) family. Different Wzz proteins confer vastly different ranges of modal lengths (4 to > 100 repeat units), despite having remarkably conserved structural folds. The molecular mechanism responsible for the selective preference for a certain number of O units is unknown. Guided by the three-dimensional structures of PCPs, we constructed a panel of chimeric molecules containing parts of two closely related Wzz proteins from Salmonella enterica and Shigella flexneri which confer different O-antigen chain length distributions. Analysis of the O-antigen length distribution imparted by each chimera revealed the region spanning amino acids 67 to 95 (region 67 to 95), region 200 to 255, and region 269 to 274 as primarily affecting the length distribution. We also showed that there is no synergy between these regions. In particular, region 269 to 274 also influenced chain length distribution mediated by two distantly related PCPs, WzzB and FepE. Furthermore, from the 3 regions uncovered in this study, region 269 to 274 appeared to be critical for the stability of the oligomeric form of Wzz, as determined by cross-linking experiments. Together, our data suggest that chain length determination depends on regions that likely contribute to stabilize a supramolecular complex.

Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of functional significance in their amino acid sequences. Here, we elucidate the topology of the Escherichia coli O157 Wzx (Wzx(EcO157)) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletion-fusions to green fluorescent protein and alkaline phosphatase. We conclude that Wzx(EcO157) consists of 12 transmembrane (TM) helices and six periplasmic and five cytosolic loops, with N and C termini facing the cytoplasm. Four TM helices (II, IV, X, and XI) contain polar residues (aspartic acid or lysine), and they may form part of a relatively hydrophilic core. Thirty-five amino acid replacements to alanine or serine were targeted to five native cysteines and most of the aspartic acid, arginine, and lysine residues. From these, only replacements of aspartic acid-85, aspartic acid-326, arginine-298, and lysine-419 resulted in a protein unable to support O-antigen production. Aspartic acid-85 and lysine-419 are located in TM helices II and XI, while arginine-298 and aspartic acid-326 are located in periplasmic and cytosolic loops 4, respectively. Further analysis revealed that the charge at these positions is required for Wzx function since conservative substitutions maintaining the same charge polarity resulted in a functional protein, whereas those reversing or eliminating polarity abolished function. We propose that the functional requirement of charged residues at both sides of the membrane and in two TM helices could be important to allow the passage of the Und-PP-linked saccharide substrate across the membrane.

Functional analysis of the C-terminal domain of the WbaP protein that mediates initiation of O antigen synthesis in Salmonella enterica

WbaP catalyzes the transfer of galactose-1-phosphate onto undecaprenyl phosphate (Und-P). The enzyme belongs to a large family of bacterial membrane proteins required for initiation of the synthesis of O antigen lipopolysaccharide and polysaccharide capsules. Previous work in our laboratory demonstrated that the last transmembrane helix and C-terminal tail region of WbaP (WbaP(CT)) are sufficient for enzymatic activity. Here, we demonstrate the cytoplasmic location of the WbaP C-terminal tail and show that WbaPCT domain N-terminally fused to thioredoxin (TrxA-WbaP(CT)) exhibits improved protein folding and enhanced transferase activity. Alanine replacement of highly conserved charged or polar amino acids identified seven critical residues for enzyme activity in vivo and in vitro. Four of these residues are located in regions predicted to be a-helical. These regions and their secondary structure predictions are conserved in distinct WbaP family members, suggesting they may contribute to form a conserved catalytic center.

An anthrax subunit vaccine candidate based on protective regions of Bacillus anthracis protective antigen and lethal factor

Studies have confirmed the key role of Bacillus anthracis protective antigen (PA) in the US and UK human anthrax vaccines. However, given the tripartite nature of the toxin, other components, including lethal factor (LF), are also likely to contribute to protection. We examined the antibody and T cell responses to PA and LF in human volunteers immunized with the UK anthrax vaccine (AVP). Individual LF domains were assessed for immunogenicity in mice when given alone or with PA. Based on the results obtained, a novel fusion protein comprising D1 of LF and the host cell-binding domain of PA (D4) was assessed for protective efficacy. Murine protection studies demonstrated that both full-length LF and D1 of LF conferred complete protection against a lethal intraperitoneal challenge with B. anthracis STI spores. Subsequent studies with the LFD1-PAD4 fusion protein showed a similar level of protection. LF is immunogenic in humans and is likely to contribute to the protection stimulated by AVP. A single vaccine comprising protective regions from LF and PA would simplify production and confer a broader spectrum of protection than that seen with PA alone.