Different heparan sulfate proteoglycans serve as cellular receptors for human papillomaviruses

Papillomaviruses replicate in stratified epithelia of skin and mucosa. Infection with certain human papillomavirus (HPV) types is the main cause of anogenital neoplasia, in particular cervical cancer. Early events of papillomavirus infectivity are poorly understood. While heparan sulfate proteoglycans (HSPGs) mediate initial binding to the cell surface, the class of proteins carrying heparan sulfates has not been defined. Here we examined two processes of papillomavirus infection, attachment of virus-like particles (VLP) to cells and infection with authentic HPV type 11 (HPV11) virions. Of the HSPGs, syndecan-1 is the major epithelial form and is strongly upregulated in wound edge keratinocytes. We employed K562 cells, which lack HSPGs except minor amounts of endogenous betaglycan, and stable clones that express cDNAs of syndecan-1, syndecan-4, or glypican-1. Binding of VLP correlated with levels of heparan sulfate on the cell surface. Parental K562 bound HPV16 VLP weakly, whereas all three K562 transfectants demonstrated enhanced binding, with the highest binding capacity observed for syndecan-1-transfected cells, which also expressed the most HSPG. For HPV11 infectivity assays, a high virion inoculum was required to infect K562 cells, whereas ectopic expression of syndecan-1 increased permissiveness eightfold and expression of syndecan-4 or glypican-1 fourfold. Infection of keratinocytes was eliminated by treatment with heparitinase, but not phospholipase C, further implicating the syndecan family of integral membrane proteins as receptor proteins. Human keratinocytes with a homozygous deletion of alpha6 integrin are permissive for HPV11 infection. These results indicate that several HSPGs can serve as HPV receptors and support a putative role for syndecan-1, rather than alpha6 integrin, as a primary receptor protein in natural HPV infection of keratinocytes.     

Syndecans serve as attachment receptors for human immunodeficiency virus type 1 on macrophages

Macrophages are thought to represent one of the first cell types in the body to be infected during the early stage of human immunodeficiency virus type 1 (HIV-1) transmission and represent a potential viral reservoir in vivo. Thus, an understanding of HIV-1 attachment to these cells is fundamental to the development of novel anti-HIV-1 therapies. Although one of the major targets of HIV-1 in vivo--CD4(+) T lymphocytes--express high CD4 levels, other major targets such as macrophages do not. We asked in this study whether this low CD4 level on macrophages is sufficient to support HIV-1 attachment to these cells or whether cell surface proteins other than CD4 are required for this process. We show that CD4 alone is not sufficient to support the initial adsorption of HIV-1 to macrophages. Importantly, we find that heparan sulfate proteoglycans (HSPGs) serve as the main class of attachment receptors for HIV-1 on macrophages. Most importantly, we demonstrate that a single family of HSPGs, the syndecans, efficiently mediates HIV-1 attachment and represents an abundant class of attachment receptors on macrophages.     

Oligosaccharide side chains of wall molecules are essential for cell-wall lysis in Chlamydomonas reinhardtii

The glycoproteins of the cell walls of Chlamydomonas are lysed during the reproductive cycle by proteases (autolysins) which are specific for their substrates. The autolysin which digests the wall of sporangia to liberate the zoospore daughter cells in the vegetative life cycle is a collagenase-like enzyme which attacks only selected domains in its wall substrates containing (hydroxy)-proline clusters. Cell-wall fractions obtained by salt-extraction (NaClO₄) and oxidizing agents (NaClO₂) and the insoluble residue were tested as substrates. The most-crosslinked insoluble inner part of the wall is the best substrate for the sporangia autolysin. Oligosaccharides obtained from the insoluble cell-wall fraction of sporangia by hydrolysis with Ba(OH)₂ inhibit autolysin action. We conclude that the oligosaccharide side chains of wall substrates are essential for forming the reactive enzyme-substrate complex.Abbreviations CSW chlorite-soluble cell-wall fraction - ICW insoluble cell-wall fraction - PSW salt-soluble fraction - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

I-domain-containing integrins serve as pilus receptors for Neisseria gonorrhoeae adherence to human epithelial cells

Two pilus receptors are identified for the pathogenic Neisseria, CD46 and complement receptor 3. An intimate association between the asialoglycoprotein receptor and gonococcal lipooligosaccharide mediates invasion of primary, male urethral epithelial cells (UECs); however, studies to identify pilus receptors on these cells have not been performed. Based on our previous studies we reasoned that the I-domain-containing (IDC), alpha(1)- and alpha(2)-integrins might serve as pilus receptors on UECs and on urethral tissue. Confocal microscopy revealed colocalization of pilus with alpha(1) and alpha(2) integrins on UECs and tissue. We found that recombinant I-domain and antibodies directed against the alpha(1)- and alpha(2)-integrins inhibited gonococcal association with UECs and with immortal cell lines of variable origin. Gonococcus-integrin colocalization occurred at early time points post infection, but this interaction dissociated with extended infection. Similarly, Western Blot analyses revealed that gonococcal pilin coimmunoprecipitates with alpha(1)- and alpha(2)-integrins. However, studies performed in parallel and that were designed to capture CD46-pilus immune complexes indicated that a CD46-pilus interaction did not occur. Collectively, these data suggest that while CD46 might be able to bind gonococcal pilus, IDC integrins are preferentially used as the initial docking site for gonococci on UECs, on urethral tissue and on some immortal cell lines.     

Characterization of epitopes of human secretory component on free secretory component, secretory IgA, and membrane-associated secretory component

We have compared the epitopes present in various forms of human secretory component by using a panel of hybridoma-derived antibodies elicited by immunizing mice with free secretory component (FSC) or secretory IgA (sIgA). Enzyme-linked immunosorbent binding assays (ELISA) were used to assess antibody binding to FSC- and SC-containing antigens, including sIgA isolated from milk, reduced and alkylated sIgA, and sIgA assembled in vitro by incubating dimeric IgA with FSC. Immunofluorescence assays were also used to assess binding to a human epithelial tumor cell line (HT29) that expresses secretory component as an integral protein of the plasma membrane. The results can be summarized as follows. 1) Most antibodies from fusions in which sIgA was the immunizing antigen bound preferentially to sIgA. 2) Most antibodies from fusions in which FSC was the immunizing antigen bound preferentially to FSC. 3) Antibodies that bound preferentially to sIgA invariably bound sIgA assembled in vitro; antibodies that bound preferentially to FSC invariably did not. 4) Antibodies that bound readily to both sIgA and FSC were rare in all fusions. 5) The monoclonal antibodies defined at least six classes of epitopes on SC, including epitopes that were a) FSC specific and reduction sensitive, b) FSC specific and reduction insensitive, c) sIgA specific and reduction-sensitive, d) sIgA specific and reduction insensitive, e) shared by FSC and sIgA and reduction-sensitive, and f) shared by FSC and sIgA and reduction-insensitive. 6) Antibodies that mediated intense immunofluorescent staining of secretory component on HT29 cell membranes were rare and constituted a distinct subset of those which recognized epitopes shared by FSC, reduced and alkylated sIgA, and some preparations of native sIgA. Results of these antibody-binding studies indicate that most SC epitopes are not shared by FSC and sIgA. Most SC-related epitopes on sIgA appear to be generated by the physical interaction of SC with dimeric IgA, whereas most epitopes on FSC are masked or altered by this interaction. Finally, epitopes that are shared by membrane SC and FSC and/or sIgA represent a minor and immunochemically distinct subset of epitopes on SC. The high proportion of unique epitopes on the different physical forms of SC suggest that the epitopes of this molecule are highly sensitive to its molecular environment. The monoclonal reagents described here will be useful in studying the structure and function of SC; quantitating FSC, sIgA, and membrane SC; purifying various molecular forms of SC by immunoaffinity chromatography; and localizing SC in human tissues and cultured cells by immunocytochemical techniques.     

The development of an immunoenzyme method for determining human secretory IgA

The results of the work on the development of an enzyme immunoassay (EIA) system for the determination of secretory IgA (S-IgA) are presented. A first, S-IgA was isolated from human colostrum and used as the basis for obtaining biologically active immunosorbent; then antibodies to S-IgA were isolated and the specific conjugate was obtained. The determination of S-IgA was carried out by the method of sandwich EIA. The newly developed EIA system permitted the determination of S-IgA only, giving no positive reactions with serum immunoglobulins. The data thus obtained make it possible to regard this assay system as specific, sensitive and suitable for further trials.     

Quantification of human urinary free secretory component, secretory and nonsecretory IgA by ELISA

The specific quantification of human urinary free secretory component (FSC), secretory IgA (SIgA) and total IgA using ELISA has been hampered by mutual interferences of these three molecules. Using affinity chromatographically purified antisera an attempt was therefore made to reduce these interferences without necessitating further assay steps. FSC and total IgA were measured in unprocessed urine by means of anti-FSC and anti-IgA as well as alkaline phosphatase-coupled anti-FSC or anti-IgA antisera. SIgA was determined using anti-IgA as well as alkaline phosphatase-coupled anti-FSC. Nonsecretory urinary IgA was calculated from the measured SIgA and total IgA. The mutual interferences of FSC, SIgA or nonsecretory IgA in the three assay systems were low and not relevant for normal samples. Normal urinary concentrations were: FSC 344 +/- (SD) 208 ng/ml (n = 120), SIgA 1,874 +/- 1,133 ng/ml (n = 123) and nonsecretory IgA, depending on the way of standardization, 712 +/- 699 (n = 56) or 878 +/- 732 ng/ml (n = 51). SIgA excretion increased with age. Lower urinary SIgA as well as total and nonsecretory IgA levels were observed in males as compared to females. No correlation evolved between the hormonal status of women and the excretion of FSC, SIgA or IgA. In IgA-deficient patients virtually no nonsecretory IgA or SIgA was detected in the urine while the FSC concentration was in the normal range.     

Annexins expressed on the cell surface serve as receptors for adhesion to immobilized fetuin-A

Fetuin-A is a major constituent of the fetal bovine serum used extensively in cell culture media. We hereby present data demonstrating that breast carcinoma cells can adhere to immobilized fetuin-A in a calcium-dependent fashion. Interestingly, the cells can also divide and attain confluency under these conditions. Using a proteomic approach, we have identified annexin-II and -VI as the putative cell surface receptors for fetuin-A in the presence of Ca2+ ions. Biotinylation of cell surface proteins followed by immunoprecipitation revealed that annexin-VI was expressed on the extracytoplasmic surface of the cell membranes. Finally, to demonstrate that annexin-II and -VI were the adhesive receptors for fetuin-A, siRNA knockdown of expression of the annexins significantly reduced the calcium-mediated adhesion. Interestingly, we demonstrated that the tumor cells could also adhere to immobilized fetuin-A in the presence of magnesium ions, and that this adhesion was most likely mediated by integrins because neutralizing antibodies against beta1 integrins substantially reduced the adhesion. Our studies suggest that the expression of annexin-II and -VI and possibly other members of the family mediate novel adhesion and signaling mechanisms in tumor cells.     

Heparan sulfate proteoglycans serve as alternative receptors for low affinity LCMV variants

Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 and thus influence viral persistence, kinetics, and cell tropism. The observation that α-DAG1 deficient cells are still highly susceptible to low affinity variants, suggests the use of an alternative receptor(s). In this study, we used a genome-wide CRISPR Cas9 knockout screen in DAG1 deficient 293T cells to identify host factors involved in α-DAG1-independent LCMV infection. By challenging cells with vesicular stomatitis virus (VSV), pseudotyped with the GP of LCMV WE HPI (VSV-GP), we identified the heparan sulfate (HS) biosynthesis pathway as an important host factor for low affinity LCMV infection. These results were confirmed by a genetic approach targeting EXTL3, a key factor in the HS biosynthesis pathway, as well as by enzymatic and chemical methods. Interestingly, a single point mutation within GP1 (S153F or Y155H) of WE HPI is sufficient for the switch from DAG1 to HS binding. Furthermore, we established a simple and reliable virus-binding assay, using directly labelled VSV-GP by intramolecular fusion of VSV-P and mWasabi, demonstrating the importance of HS for virus attachment but not entry in Burkitt lymphoma cells after reconstitution of HS expression. Collectively, our study highlights the essential role of HS for low affinity LCMV infection in contrast to their high affinity counterparts. Residual LCMV infection in double knockouts indicate the use of (a) still unknown entry receptor(s).     

G protein-coupled receptors serve as mechanosensors for fluid shear stress in neutrophils

Many cells respond to fluid shear stress but in a cell type-specific fashion. Fluid shear stress applied to leukocytes serves to control pseudopod formation, migration, and other functions. Specifically, fresh neutrophils or neutrophilic leukocytes derived from differentiated HL60 cells respond to fluid shear stress by cytoplasmic pseudopod retraction. The membrane elements that sense fluid shear and induce such a specific response are still unknown, however. We hypothesized that membrane receptors may serve as fluid shear sensors. We found that fluid shear decreased the constitutive activity of G protein-coupled receptors (GPCRs). Inhibition of GPCR constitutive activity by inverse agonists abolished fluid shear stress-induced cell area reduction. Among the GPCRs in neutrophils, the formyl peptide receptor (FPR) exhibits relatively high constitutive activity. Undifferentiated HL60 cells that lacked FPR formed few pseudopods and showed no detectable response to fluid shear stress, whereas expression of FPR in undifferentiated HL60 cells caused pseudopod projection and robust pseudopod retraction during fluid shear. FPR small interfering RNA-transfected differentiated HL60 cells exhibited no response to fluid shear stress. These results suggest that GPCRs serve as mechanosensors for fluid shear stress in neutrophils by decreasing its constitutive activity and reducing pseudopod projection. leukocyte; constitutive activity; mechanotransduction; formyl peptide receptor     

Dislocation of ricin toxin A chains in human cells utilizes selective cellular factors

Ricin is a potent A-B toxin that is transported from the cell surface to the cytosol, where it inactivates ribosomes, leading to cell death. Ricin enters cells via endocytosis, where only a minute number of ricin molecules reach the endoplasmic reticulum (ER) lumen. Subsequently, the ricin A chain traverses the ER bilayer by a process referred to as dislocation or retrograde translocation to gain access to the cytosol. To study the molecular processes of ricin A chain dislocation, we have established, for the first time, a human cell system in which enzymatically attenuated ricin toxin A chains (RTA(E177D) and RTA(Δ177-181)) are expressed in the cell and directed to the ER. Using this human cell-based system, we found that ricin A chains underwent a rapid dislocation event that was quite distinct from the dislocation of a canonical ER soluble misfolded protein, null Hong Kong variant of α(1)-antitrypsin. Remarkably, ricin A chain dislocation occurred via a membrane-integrated intermediate and utilized the ER protein SEL1L for transport across the ER bilayer to inhibit protein synthesis. The data support a model in which ricin A chain dislocation occurs via a novel strategy of utilizing the hydrophobic nature of the ER membrane and selective ER components to gain access to the cytosol.     

Fc receptors for IgA on human B, and human non-B, non-T lymphocytes.

Recently, receptors for IgA were demonstrated on subpopulations of human T lymphocytes. In this report, TNP-modified ox erythrocytes coated with the IgA myeloma MOPC-315 were used to detect IgA receptor-bearing lymphocytes within the human non T cell lymphocyte population. A mean of 5.3% (range 2.9 to 12.4%) of E-rosette negative human lymphocytes bound IgA-coated indicator cells. Blocking studies with soluble IgA, IgG, and IgM demonstrated that the IgA receptors on the non-T cell populations were separate and distinct from the Fc-receptors for IgG and IgM. Fractionation of the non-T lymphocytes on anti-human (Fab)2 columns into sIg+ and sIg- populations or by rosetting with EAC to provide CRL+ and CRL- populations demonstrated that Fc-IgA receptors were present on a subpopulation of sIg+, CRL+ lymphocytes, and also on sIg- (non-T, non-B) lymphocytes.     

Degradation of human secretory IgA1 and IgA2 by Entamoeba histolytica surface-associated proteolytic activity

The protozoan Entamoeba histolytica is the etiological agent of amebiasis, an infection with high prevalence worldwide. The host-ameba relationship outcome depends on parasite and host factors, and among these is secretory IgA. These antibodies reduce mucosal colonization by pathogens and neutralize a variety of toxins and enzymes. The functionality of secretory IgA depends on its integrity. Some bacteria produce IgA proteases that cleave mainly the IgA1 subclass; live E. histolytica trophozoites, and other ameba fractions are also able to degrade human IgA. The aim of this study was to determine if serum and secretory IgA, its subclasses and secretory component, are degraded by cysteine proteases, which are present and active on the surface of glutaraldehyde-fixed amebas. It was observed that secretory IgA1, IgA2, free and IgA-bound secretory component were degraded by E. histolytica surface-associated cysteine proteinases. Secretory IgA2, although it was degraded, conserved its ability to agglutinate live amebas better than IgA1. Therefore, while specificity of known ameba cysteine proteases is cathepsin B-like and is different from bacterial IgA proteases, IgA2 was functionally more resistant than IgA1 to ameba surface-associated cysteine protease degradation, similar to the greater resistance of IgA2 to bacterial IgA-specific proteases.     

Disulfide bonding of secretory component to a single monomer subunit in human secretory IgA.

The arrangement of disulfide bonds joining secretory component (SC) to the alpha chains in secretory IgA was studied by determining the molecular size of the principal fragments resulting from CNBr digestion of secretory dimeric Fc fragments from IgA (Fc)2alpha fragments). In vitro complexes formed by incubating 125I-free SC and myeloma 131I-(Fc)2alpha fragments were isolated by gel filtration and subsequently digested with cyanogen bromide. The CNBr digests of SC-(Fc)2alpha fragments were analyzed by gel filtration in 5 M guanidine. Two principal fragments were obtained, one containing a monomeric Fc fragment from IgA (Fcalpha) associated with SC (m.w. congruent to 110,000) and a second containing the second Fcalpha monomer (m.w. congruent to 50,000) from the dimeric SC-(Fc)2alpha. Similar results were obtained when secretory (Fc)2alpha fragments isolated from native secretory IgA dimer were subjected to CNBr digestion. The data indicate that SC is disulfide bonded to a single monomer subunit in secretory IgA dimer.     

Dihydroquinolines with amine-containing side chains as potent n-NOS inhibitors

Dihydroquinolines with aminoalkyl side chains have been synthesized and have been shown to be potent n-NOS inhibitors. A marked selectivity versus e-NOS of up to approximately 300-fold was observed, whereas i-NOS was moderately inhibited.     

Constructing side chains on near-native main chains for ab initio protein structure prediction

Is there value in constructing side chains while searching protein conformational space during an ab initio simulation? If so, what is the most computationally efficient method for constructing these side chains? To answer these questions, four published approaches were used to construct side chain conformations on a range of near-native main chains generated by ab initio protein structure prediction methods. The accuracy of these approaches was compared with a naive approach that selects the most frequently observed rotamer for a given amino acid to construct side chains. An all-atom conditional probability discriminatory function is useful at selecting conformations with overall low all-atom root mean square deviation (r.m.s.d.) and the discrimination improves on sets that are closer to the native conformation. In addition, the naive approach performs as well as more sophisticated methods in terms of the percentage of chi(1) angles built accurately and the all-atom r. m.s.d., between the native and near-native conformations. The results suggest that the naive method would be extremely useful for fast and efficient side chain construction on vast numbers of conformations for ab initio prediction of protein structure.