Functional properties of complement factor H-related proteins FHR-3 and FHR-4: binding to the C3d region of C3b and differential regulation by heparin

The human factor H-related proteins FHR-3 and FHR4 are members of a family of proteins related to the complement factor H. Here, we report that the two proteins bind to the C3d region of complement C3b. The apparent K(A) values for the interactions of FHR-3 and FHR-4 with C3b are 7.5 x 10(6) M(-1) and 2.9 x 10(6) M(-1), respectively. Binding studies performed with C3b-coated pneumococci confirmed the results obtained with the biosensor system. A C-terminal construct of factor H showed similar binding characteristics. The interaction of FHR-3, but not of FHR4, with opsonised pneumococci was inhibited by heparin.     

Human factor H-related protein 5 has cofactor activity, inhibits C3 convertase activity, binds heparin and C-reactive protein, and associates with lipoprotein

Factor H-related protein 5 (FHR-5) is a recently discovered member of the factor H (fH)-related protein family. FHR proteins are structurally similar to the complement regulator fH, but their biological functions remain poorly defined. FHR-5 is synthesized in the liver and consists of 9 short consensus repeats (SCRs), which display various degrees of homology to those of fH and the other FHR proteins. FHR-5 colocalizes with complement deposits in vivo and binds C3b in vitro, suggesting a role in complement regulation or localization. The current study examined whether rFHR-5 exhibits properties similar to those of fH, including heparin binding, CRP binding, cofactor activity for the factor I-mediated degradation of C3b and decay acceleration of the C3 convertase. rFHR-5 bound heparin-BSA and heparin-agarose and a defined series of truncations expressed in Pichia pastoris localized the heparin-binding region to within SCRs 5-7. rFHR-5 bound CRP, and this binding was also localized to SCRs 5-7. FHR-5 inhibited alternative pathway C3 convertase activity in a fluid phase assay; however, dissociation of the convertase was not observed in a solid phase assay. rFHR-5 displayed factor I-dependent cofactor activity for C3b cleavage, although it was apparently less effective than fH. In addition, we demonstrate association of FHR-5 with high density lipid lipoprotein complexes in human plasma. These results demonstrate that FHR-5 shares properties of heparin and CRP binding and lipoprotein association with one or more of the other FHRs but is unique among this family of proteins in possessing independent complement-regulatory activity.     

Homology modelling of the human eukaryotic initiation factor 5A (eIF-5A).

Homology modelling of the human eIF-5A protein has been performed by using a multiple predictions strategy. As the sequence identity between the target and the template proteins is nearly 30%, which is lower than the commonly used threshold to apply with confidence the homology modelling method, we developed a specific predictive scheme by combining different sequence analyses and predictions, as well as model validation by comparison to structural experimental information. The target sequence has been used to find homologues within sequence databases and a multiple alignment has been created. Secondary structure for each single protein has been predicted and compared on the basis of the multiple sequence alignment, in order to evaluate and adjust carefully any gap. Therefore, comparative modelling has been applied to create the model of the protein on the basis of the optimized sequence alignment. The quality of the model has been checked by computational methods and the structural features have been compared to experimental information, giving us a good validation of the reliability of the model and its correspondence to the protein structure in solution. Last, the model was deposited in the Protein Data Bank to be accessible for studies on the structure-function relationships of the human eIF-5A.     

A SECIS binding protein (SBP) is distinct from selenocysteyl-tRNA protecting factor (SePF).

In mammals, most of the selenium contained in their body is present as an unusual amino acid, selenocysteine (Sec), whose codon is UGA. Because the UGA codon is normally recognized as a translational stop signal, it is intriguing how cells recognize and distinguish the UGA Sec codon from the UGA stop codon. In eukaryotic selenoprotein mRNAs, it has been proposed that a conserved stem-loop structure designated Sec insertion sequence (SECIS) located in the 3'-untranslated regions is required for recognition of UGA as a Sec codon. Although some proteins (SBPs) have been reported to bind to SECIS, it is not clear how the SECIS element can mediate Sec insertion at UGA. Eukaryotic Sec-tRNA(Sec) is not recognized by elongation factor EF-1alpha, but is recognized specifically by a Sec-tRNA(Sec) protecting factor, SePF, in bovine liver extracts. In this study, we provide evidence that SePF is distinct from SBP by chromatography. Upon UV irradiation, the SECIS RNA was cross-linked to a 47.5 kDa protein, a likely candidate of SBP, that is contained in the complex with a molecular mass of 150 kDa. These results suggest that SBP and SePF play different roles for the Sec incorporation. To our knowledge, this is the first demonstration that SBP is discriminated from the factor which directly recognizes Sec-tRNA(Sec), providing a novel clue to the mechanism of selenocysteine decoding in eukaryotes.     

Chemotactic factor inactivator interaction with Gc-globulin (vitamin D-binding protein). A mechanism of modulating the chemotactic activity of C5a

Chemotactic factor inactivator (CFI) can decrease the neutrophil chemotactic activity of C5a. Gc-Globulin (GcG) can function as a cochemotaxin for C5a by binding to C5a or C5a des Arg and enhancing its chemotactic potency. We hypothesized that CFI might interact with GcG and thus decrease the chemotactic activity of C5a. CFI was found to markedly inhibit the neutrophil chemotactic activity of partially purified C5a containing GcG (p less than 0.01). Addition of GcG was able to reverse the capacity of CFI to inhibit C5a-directed neutrophil chemotaxis (p less than 0.01). CFI had no significant effect on neutrophil chemotaxis when incubated with C5a depleted of GcG or C5a des Arg. CFI was also able to inhibit the interaction of C5a with GcG adsorbed to plastic. To determine if CFI interacted with GcG, a sandwich ELISA was used. These ELISA tests demonstrated that CFI directly interacted with GcG in a dose-dependent manner that was both heat and pH sensitive. To investigate the possibility of enzymatic degradation of C5a by CFI, CFI preparations were analyzed for carboxypeptidase activity, aminopeptidase activity, and for the capacity to cleave dansylated C5a. No enzymatic activity or cleavage was observed. Furthermore, the direct interaction of CFI with C5a and C5a des Arg was assessed by ELISA tests and column chromatography and no interaction was observed. These results suggest that CFI modulates C5a-directed neutrophil chemotaxis by interacting with GcG and preventing GcG from enhancing the chemotactic potency of C5a.     

Human placenta S-adenosylmethionine: protein carboxyl O-methyltransferase (protein methylase II). Purification and characterization

1. Protein methylase II was purified from human placenta approx. 8700-fold with a yield of 14%. 2. Unlike protein methylase II from other sources, the activity of human placenta enzyme was completely inhibited by 2 mM Cu2+. Other divalent ions were without effect. 3. Human chorionic gonadotropin (HCG), immunoglobulin A and calf thymus histones served as good in vitro substrates for the enzyme, particularly HCG. 4. The Km for S-adenosyl-L-methionine and Ki for S-adenosyl-L-homocysteine were 2.08 x 10(-6) and 5.8 x 10(-7) M, respectively. 5. The protein methylase II activity in human placenta changed with gestational age, the activity at 1st and 2nd trimester being approximately twice that of term placenta.     

人乳头瘤病毒E5蛋白

E5是人乳头瘤病毒(human papillomavirus, HPV)表达的3个致瘤蛋白之一,它的致瘤能力较弱,在一些HPV相关的恶性肿痛经常缺乏E5表达.E5主要通过与表皮生长因子受体(epidermal growth factor receptor,EGFR)和V型H+-腺三磷酶(ATPase)等的相互作用,在肿瘤形成早期对细胞增殖、转化、免疫逃避等细胞行为发挥重要影响,促进宫颈上皮从HPV感染到恶性转变.     

The human factor H-related gene 2 (FHR2): structure and linkage to the coagulation factor XIIIb gene

The human factor H-related gene 2 (FHR2) encodes a serum protein structurally and immunologically related to complement factor H. We describe the isolation and genomic organization of the human FHR2 gene from a yeast artificial chromosome library. The FHR2 gene is organized in five exosn and span about 7 kilobases (kb) of human genomic DNA. A comparison with the corresponding cDNA sequence (clone DDESK59) shows that the analyzed FHR2 gene has a deleted region within exon 4. A new splice acceptor site created in the truncated exon indicates that the analyzed gene could be translated to a truncated protein. Further, we demonstrate that the genes for FHR2 and subunit of coagulation factor XIII are located in the same 165 kb YAC DNA. Thus, the three structurally related genes FXIIIb, FHR2, and factor H are linked on human chromosome 1 in the regulators of complement activiation (RCA) gene cluster. The physical linkage of the FHR2 and the factor H genes provides additional evidence for a close relatedness of complement factor H and the factor H-related proteins. The linkage and the almost exclusive organization in short consensus repeat-containing domains indicates a close evolutionary relationship of the FXIIIb, FHR2, and factor H genes.The nucleotide sequence data reported in this paper have been submitted to the EMBL and GenBank nucleotide sequence databases and have been assigned the accession number X86564 (exon 1), X86565 (exon 2), X86566 (exon 3 and 4), and X86567 (exon 5)     

Association of a thioredoxin-like protein with chloroplaast coupling factor (CF1).

The δ subunit isolated from chloroplast coupling factor (CF₁) preparations partially replaced thioredoxin in the dithiothreitol-linked activation of chloroplast fructose 1,6-bisphosphatase. The δ subunit fraction also stimulated the dithiothreitol-dependent ATPase of heated CF₁ in a manner analogous to that obseryed with each of the three thioredoxins isolated from spinach leaves (thioredoxins f, m, and c). The δ subunit used in most of these experiments was obtained from CF₁ that had been isolated by a newly devised procedure based on acid precipitation.     

Genetic interactions of yeast eukaryotic translation initiation factor 5A (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling

The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIF5A domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIF5A may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes.     

Complement factor H-related proteins CFHR2 and CFHR5 represent novel ligands for the infection-associated CRASP proteins of Borrelia burgdorferi

Background

One virulence property of Borrelia burgdorferi is its resistance to innate immunity, in particular to complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASP) which interact with complement regulator factor H (CFH) and factor H-like protein 1 (FHL1) or factor H-related protein 1 (CFHR1). In the present study we elucidate the role of the infection-associated CRASP-3 and CRASP-5 protein to serve as ligands for additional complement regulatory proteins as well as for complement resistance of B. burgdorferi.

Methodology/Principal Findings

To elucidate whether CRASP-5 and CRASP-3 interact with various human proteins, both borrelial proteins were immobilized on magnetic beads. Following incubation with human serum, bound proteins were eluted and separated by Glycine-SDS-PAGE. In addition to CFH and CFHR1, complement regulators CFHR2 and CFHR5 were identified as novel ligands for both borrelial proteins by employing MALDI-TOF. To further assess the contributions of CRASP-3 and CRASP-5 to complement resistance, a serum-sensitive B. garinii strain G1 which lacks all CFH-binding proteins was used as a valuable model for functional analyses. Both CRASPs expressed on the B. garinii outer surface bound CFH as well as CFHR1 and CFHR2 in ELISA. In contrast, live B. garinii bound CFHR1, CFHR2, and CFHR5 and only miniscute amounts of CFH as demonstrated by serum adsorption assays and FACS analyses. Further functional analysis revealed that upon NHS incubation, CRASP-3 or CRASP-5 expressing borreliae were killed by complement.

Conclusions/Significance

In the absence of CFH and the presence of CFHR1, CFHR2 and CFHR5, assembly and integration of the membrane attack complex was not efficiently inhibited indicating that CFH in co-operation with CFHR1, CFHR2 and CFHR5 supports complement evasion of B. burgdorferi.     

Molecular expression of the negative growth factor murine beta-galactoside binding protein (mGBP).

Characterisation of the negative growth factor mGBP at molecular and biological levels indicates that the protein has no lectin nature and suggests instead a participation in the cytokine network. The protein is shown to be expressed as a monomer in two forms, one of which is non-covalently linked to a glycan complex. This confers greater efficiency to the inhibitor and may favour a paracrine role. The two monomeric forms may oxidise into tetramers which retain biological activity, but lack ability to link to specific saccharide residues.