The L1 major capsid protein of human papillomavirus (HPV) type 11, a 55-kDa polypeptide, forms particulate structures resembling native virus with an average particle diameter of 50 -60 nm when expressed in the yeast Saccharomyces cerevisiae. We show in this report that these virus-like particles (VLPs) interact with heparin and with cell-surface glycosaminoglycans (GAGs) resembling heparin on keratinocytes and Chinese hamster ovary cells. The binding of VLPs to heparin is shown to exhibit an affinity comparable to that of other identified heparin-binding proteins. Immobilized heparin chromatography and surface plasmon resonance were used to show that this interaction can be specifically inhibited by free heparin and dextran sulfate and that the effectiveness of the inhibitor is related to its molecular weight and charge density. Sequence comparison of nine human L1 types revealed a conserved region of the carboxyl terminus containing clustered basic amino acids that bear resemblance to proposed heparin-binding motifs in unrelated proteins. Specific enzymatic cleavage of this region eliminated binding to both immobilized heparin and human keratinocyte (HaCaT) cells. Removal of heparan sulfate GAGs on keratinocytes by treatment with heparinase or heparitinase resulted in an 80 -90% reduction of VLP binding, whereas treatment of cells with laminin, a substrate for ␣ 6 integrin receptors, provided minimal inhibition. Cells treated with chlorate or substituted ␤-D-xylosides, resulting in undersulfation or secretion of GAG chains, also showed a reduced affinity for VLPs. Similarly, binding of VLPs to a Chinese hamster ovary cell mutant deficient in GAG synthesis was shown to be only 10% that observed for wild type cells. This report establishes for the first time that the carboxyl-terminal portion of HPV L1 interacts with heparin, and that this region appears to be crucial for interaction with the cell surface.

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The majority of human urinary stones are primarily composed of calcium salts. Although normal urine is frequently supersaturated with respect to calcium oxalate, most humans do not form stones. Inhibitors are among the multiple factors that may influence the complex process of urinary stone formation. We have isolated an inhibitor of calcium oxalate crystal growth from human urine by monoclonal antibody immunoaffinity chromatography. The N-terminal amino acid sequence and acidic amino acid content of this aspartic acid-rich protein, uropontin, are similar to those of other pontin proteins from bone, plasma, breast milk, and cells. The inhibitory effect of uropontin on calcium oxalate crystal growth in vitro supports the concept that pontins may have a regulatory role. This function would be analogous to that of other members of the aspartic acid-rich protein superfamily, which stereospecifically regulate the mineralization fronts of calcium-containing crystals.Urinary tract stone disease is a common human malady, and the vast majority of stones formed in the urinary space are mineralized with calcium salts (1, 2). The elements contained in urine also provide a potential model system for evaluating the biologic control of mineralization in other body fluids. Although normal urine is frequently supersaturated with respect to calcium oxalate, most humans do not form stones. Urinary stone formation is a complex process involving multiple factors, and the precise role of the inhibitors that are present within urine is uncertain. The majority of the inhibition of crystal growth observed in normal urine is due to the presence of protein macromolecules rather than to the presence of lower molecular weight molecules (3). We approached the problem of identifying other crystal inhibitor proteins by preparing monoclonal antibodies from rats immunized with the main inhibitory peak of human urine protein (3). One of these monoclonal antibodies was used to purify an inhibitor of calcium oxalate crystal growth from human urine by immunoaffinity chromatography. METHODS Protein Purification. Human urine samples were carried through all procedures in the presence of 0.02% sodium azide and two protease inhibitors, 0.5 mM phenylmethanesulfonyl fluoride and 1.0 mM N-ethylmaleimide, and were partially depleted of the most abundant protein in normal urine, Tamm-Horsfall protein (TH), by salt precipitation followed by centrifugation at 5000 x g for 30 min (4). TH-depleted urine was adsorbed to DEAE-cellulose, batch eluted, and fractionated by DEAE-cellulose column chromatography, using a 0.1-0.4 M NaCl linear gradient in Tris buffer (3). The TH depletion step was performed since we found that TH is present within the main inhibitory peak from DEAE-cellulose and does not inhibit crystal growth in the assay used in the present study (5). Inhibitory activity of fractions was assayed by measuring the inhibition of incorporation of [14C]oxalate (Amersham) into calcium oxalate monohydrate seed crystals from a metastable calcium oxalat...

We have found correlations between rate constants and the difference in redox potential of the reactants for electron-transfer reactions between oxidized cytochromes and either photoproduced riboflavin or flavin mononucleotide (FMN) semiquinones (the latter rate constants extrapolated to infinite ionic strength). The riboflavin-cytochrome rate constants are about 70% of those for reduction by lumiflavin, probably because of steric interference by the ribityl side chain. Reduction of cytochromes by FMN semiquinone was ionic strength dependent in all cases, due to electrostatic interactions. Extrapolation of rate constants to infinite ionic strength shows that the phosphate exerts a significant steric effect as well (rate constants average about 27% of those for lumiflavin, although part of this decrease is due to a difference in the semiquinone pK value). Differences in the magnitude of the FMN steric effect correlate well with surface topology differences for those cytochromes whose three-dimensional structures are known. Mitochondrial cytochromes c and the cytochromes c2 all showed attractive (plus-minus) interaction with FMN in spite of the fact that some of these proteins have large net negative charges. Four small c-type cytochromes (including Pseudomonas cytochrome c-551) show a weak repulsive interaction with FMN semiquinone. We conclude that flavosemiquinones interact at a site on the cytochromes that is near the exposed heme edge. There is a large positive electrostatic field at this site in mitochondrial cytochrome c and the cytochromes c2, but this region is primarily hydrophobic in Pseudomonas cytochrome c-551 and in the other small bacterial cytochromes.(ABSTRACT TRUNCATED AT 250 WORDS)

The development of protein subunit vaccines to combat some of the world's deadliest pathogens such as a malaria parasite, Plasmodium falciparum, is stalled, due in part to the inability to induce and sustain high-titer antibody responses. Here, we show the induction of persistent, high-titer antibody responses to recombinant Pfs25H, a human malarial transmission-blocking protein vaccine candidate, after chemical conjugation to the outer-membrane protein complex (OMPC) of Neisseria meningitidis serogroup B and adsorption to aluminum hydroxyphosphate. In mice, the Pfs25H-OMPC conjugate vaccine was >1,000 times more potent in generating anti-Pfs25H ELISA reactivity than a similar 0.5-g dose of Pfs25H alone in Montanide ISA720, a water-in-oil adjuvant. The immune enhancement requires covalent conjugation between Pfs25H and the OMPC, given that physically mixed Pfs25H and OMPC on aluminum hydroxyphosphate failed to induce greater activity than the nonconjugated Pfs25H on aluminum hydroxyphosphate. The conjugate vaccine Pfs25H-OMPC also was highly immunogenic in rabbits and rhesus monkeys. In rhesus monkeys, the antibody responses were sustained over 18 months, at which time another vaccination with nonconjugated Pfs25H induced strong anamnestic responses. The vaccine-induced anti-Pfs25-specific antibodies in all animal species blocked the transmission of parasites to mosquitoes. Protein antigen conjugation to OMPC or other protein carrier may have general application to a spectrum of protein subunit vaccines to increase immunogenicity without the need for potentially reactogenic adjuvants. malaria ͉ Pfs25 ͉ transmission-blocking vaccine A s reported by the World Health Organization in 2004, the worldwide incidence of malaria is Ϸ300 million clinical cases and 1.3 million deaths annually (1). Of the four species of malaria parasites that infect humans, Plasmodium falciparum is responsible for the majority of these deaths, and Plasmodium vivax accounts for Ͼ50% of all malarial infections outside Africa and 10% of those in Africa. Mounting drug resistance by the malaria parasite makes chemotherapy increasingly difficult. Three types of malarial vaccines are under research and development: (i) vaccines targeting the liver-stage parasite development for sterile immunity; (ii) vaccines targeting the blood-stage parasite to reduce disease burden; and (iii) vaccines targeting the parasite development in the mosquito stage to block transmission, called transmission-blocking vaccines (TBVs). For all three types of vaccines, antibody is important. For TBVs, antibody is the only mechanism for providing immune protection. TBVelicited functional antibodies, ingested with the sexual stages of the parasite in a blood meal by a mosquito, will inhibit or block parasite development in the mosquito.Pxs25 proteins encoded by orthologous genes and expressed on the surface of zygotes and ookinetes during the development of the malaria parasite P. falciparum (Pfs25) and P. vivax (Pvs25) are leading candidates for mosquito-stage transmission...