Evaluation of hepatitis C virus glycoprotein E2 for vaccine design: an endoplasmic reticulum-retained recombinant protein is superior to secreted recombinant protein and DNA-based vaccine candidates.

Hepatitis C virus (HCV) is the leading causative agent of blood-borne chronic hepatitis and is the target of intensive vaccine research. The virus genome encodes a number of structural and nonstructural antigens which could be used in a subunit vaccine. The HCV envelope glycoprotein E2 has recently been shown to bind CD81 on human cells and therefore is a prime candidate for inclusion in any such vaccine. The experiments presented here assessed the optimal form of HCV E2 antigen from the perspective of antibody generation. The quality of recombinant E2 protein was evaluated by both the capacity to bind its putative receptor CD81 on human cells and the ability to elicit antibodies that inhibited this binding (NOB antibodies). We show that truncated E2 proteins expressed in mammalian cells bind with high efficiency to human cells and elicit NOB antibodies in guinea pigs only when purified from the core-glycosylated intracellular fraction, whereas the complex-glycosylated secreted fraction does not bind and elicits no NOB antibodies. We also show that carbohydrate moieties are not necessary for E2 binding to human cells and that only the monomeric nonaggregated fraction can bind to CD81. Moreover, comparing recombinant intracellular E2 protein to several E2-encoding DNA vaccines in mice, we found that protein immunization is superior to DNA in both the quantity and quality of the antibody response elicited. Together, our data suggest that to elicit antibodies aimed at blocking HCV binding to CD81 on human cells, the antigen of choice is a mammalian cell-expressed, monomeric E2 protein purified from the intracellular fraction.

Innate defenses in the liver during Listeria infection.

The majority of pathogens entering the bloodstream are cleared by the liver. Listeria monocytogenes, an important natural pathogen of humans, is a useful tool for examining protective immune responses during systemic infections of mice. Innate immunity contributes to blood clearance and eventual sterilization of the liver subsequent to Listeria infections. Effector mechanisms expressed in the liver early after infections are orchestrated by complex interactions between resident populations, i.e. hepatocytes and Kupffer cells, with infiltrating monocytes, neutrophils, and natural killer cells. These interactions include cell to cell contact through adhesion molecules, as well as communication through secretion of cytokines and chemokines. The liver environment, as the interface between blood-borne pathogens and innate host defenses, is reviewed here.

Interferon alpha/beta-mediated inhibition and promotion of interferon gamma: STAT1 resolves a paradox.

Induction of high systemic levels of type 1 interferons (IFNs) IFN-alpha and IFN-beta is a hallmark of many viral infections. In addition to their potent antiviral effects, these cytokines mediate a number of immunoregulatory functions and can promote IFN-gamma expression in T cells. However, during viral infections of mice IFN-gamma production is not always observed at the same time as systemic IFN-alpha/beta production and when, elicited at these times, is IFN-alpha/beta-independent. We demonstrate that type 1 interferons not only fail to induce, but also act to inhibit, IFN-gamma expression by both NK and T cells. The mechanism of inhibition is dependent upon the IFN-alpha/beta receptor and the signal transducer and activator of transcription 1 (STAT1). In the absence of STAT1, not only are the IFN-alpha/beta-mediated inhibitory effects completely abrogated, but the cytokines themselves can induce IFN-gamma expression. These results indicate that endogenous biochemical pathways are in place to negatively regulate NK and T cell IFN-gamma expression elicited by IFN-alpha/beta or other stimuli, at times of innate responses to viral infections. They also show that type 1 interferon signaling can occur through STAT1-dependent and independent mechanisms and suggest that efficient induction of IFN-gamma expression by IFN-alpha/beta requires STAT1 regulation. Such immunoregulatory pathways may be critical for shaping the endogenous innate and virus-specific adaptive immune responses to viral infections.

Breast tumor characteristics as predictors of mammographic detection: comparison of interval- and screen-detected cancers.

BACKGROUND: Although mammographic screening is useful for detecting early breast cancer, some tumors are detected in the interval between screening examinations. This study attempted to characterize fully the tumors detected in the two different manners. METHODS: Our study utilized a case-control design and involved a cohort of women undergoing mammographic screening within the defined population of a health maintenance organization (the Group Health Cooperative of Puget Sound). Women were classified as having "interval" or "interval-detected" cancers (n = 150) if their diagnosis was made within 24 months after a negative-screening mammogram or one that indicated a benign condition. Cancers were classified as "screen detected" (n = 279) if the diagnosis occurred after a positive assessment by screening mammography. Tumors from women in each group were evaluated for clinical presentation, histology, proliferative characteristics, and expression of hormone receptors, p53 tumor suppressor protein, and c-erbB-2 protein. RESULTS: Interval-detected cancers occurred more in younger women and were of larger tumor size than screen-detected cancers. In unconditional logistic regression models adjusted for age and tumor size, tumors with lobular (odds ratio [OR] = 1.9; 95% confidence interval [CI] = 0.9-4.2) or mucinous (OR = 5.5; 95% CI = 1.5-19.4) histology, high proliferation (by either mitotic count [OR = 2.9; 95% CI = 1.5-5.7] or Ki-67 antigen expression [OR = 2.3; 95% CI = 1.3-4.1]), high histologic grade (OR = 2.1; 95% CI = 1.2-4.0), high nuclear grade (OR = 2.0; 95% CI = 1.0-3.7), or negative estrogen receptor status (OR = 1.8; 95% CI = 1.0-3.1) were more likely to surface in the interval between screening examinations. Tumors with tubular histology (OR = 0.2; 95% CI = 0.0-0.8) or with a high percentage of in situ components (50%) (OR = 0.5; 95% CI = 0.2-1.2) were associated with an increased likelihood of screen detection. CONCLUSIONS: Our data from a large group of women in a defined population indicate that screening mammography may miss tumors of lobular or mucinous histology and some rapidly proliferating, high-grade tumors.

Natural killer cells in antiviral defense: function and regulation by innate cytokines.

Natural killer (NK) cells are populations of lymphocytes that can be activated to mediate significant levels of cytotoxic activity and produce high levels of certain cytokines and chemokines. NK cells respond to and are important in defense against a number of different infectious agents. The first indications for this function came from the observations that virus-induced interferons alpha/beta (IFN-alpha and -beta) are potent inducers of NK cell-mediated cytotoxicity, and that NK cells are important contributors to innate defense against viral infections. In addition to IFN-alpha/beta, a wide range of other innate cytokines can mediate biological functions regulating the NK cell responses of cytotoxicity, proliferation, and gamma interferon (IFN-gamma) production. Certain, but not all, viral infections induce interleukin 12 (IL-12) to elicit NK cell IFN-gamma production and antiviral mechanisms. However, high levels of IFN-alpha/beta appear to be unique and/or uniquely dominant in the context of viral infections and act to regulate other innate responses, including induction of NK cell proliferation in vivo and overall negative regulation of IL-12 production. A detailed picture is developing of particular innate cytokines activating NK cell responses and their consorted effects in providing unique endogenous milieus promoting downstream adaptive responses, most beneficial in defense against viral infections.

Two roads diverged: interferon alpha/beta- and interleukin 12-mediated pathways in promoting T cell interferon gamma responses during viral infection.

Viral infections induce CD8 T cell expansion and interferon (IFN)-gamma production for defense, but the innate cytokines shaping these responses have not been identified. Although interleukin (IL)-12 has the potential to contribute, IL-12-dependent T cell IFN-gamma has not been detected during viral infections. Moreover, certain viruses fail to induce IL-12, and elicit high levels of IFN-alpha/beta to negatively regulate it. The endogenous factors promoting virus-induced T cell IFN-gamma production were defined in studies evaluating CD8 T cell responses during lymphocytic choriomeningitis virus infections of mice. Two divergent supporting pathways were characterized. Under normal conditions of infections, the CD8 T cell IFN-gamma response was dependent on endogenous IFN-alpha/beta effects, but was IL-12 independent. In contrast, in the absence of IFN-alpha/beta functions, an IL-12 response was revealed and substituted an alternative pathway to IFN-gamma. IFN-alpha/beta-mediated effects resulted in enhanced, but the alternative pathway also promoted, resistance to infection. These observations define uniquely important IFN-alpha/beta-controlled pathways shaping T cell responses during viral infections, and demonstrate plasticity of immune responses in accessing divergent innate mechanisms to achieve similar ultimate goals.

Activation of the grp78 and grp94 promoters by hepatitis C virus E2 envelope protein.

The hepatitis C virus E1 and E2 envelope proteins are targeted to the endoplasmic reticulum, but instead of being secreted, they are retained in a pre-Golgi compartment, at least partly in a misfolded state. Since secretory proteins which are retained in the endoplasmic reticulum frequently can activate the transcription of intraluminal chaperone proteins, we measured the effect of the E1 and E2 proteins on the promoters of two such chaperones, GRP78 (BiP) and GRP94. We found that E2 but not E1 protein activates these two promoters, as assayed by a reporter gene system. Furthermore, E2 but not E1 protein induces the synthesis of GRP78 from the endogenous cellular gene. We also found that E2 but not E1 protein expressed in mammalian cells is bound tightly to GRP78. This association may explain the ability of E2 protein to activate transcription, since GRP78 has been postulated to be a sensor of stress in the endoplasmic reticulum. Since overexpression of GRP78 has been shown to decrease the sensitivity of cells to killing by cytotoxic T lymphocytes and to increase tumorigenicity and resistance to antitumor drugs, this activity of E2 protein may be involved in the pathogenesis of hepatitis C virus-induced diseases.

CD4+ and CD8+ T cell interactions in IFN-gamma and IL-4 responses to viral infections: requirements for IL-2.

Cytokine responses to lymphocytic choriomeningitis virus infections were evaluated, and CD8+ T cell, CD4+ T cell, and IL-2 contributions delineated. In immunocompetent mice, lymphocytic choriomeningitis virus induced both IFN-gamma and IL-4 as well as IL-2. Experiments in mice either beta2-microglobulin-deficient, lacking MHC class I molecules and CD8+ T cells, or A beta(b)-deficient, lacking MHC class II molecules and CD4+ T cells, demonstrated that mixtures of T cell responses were required for optimal ex vivo cytokine productions. Intracellular cytokine expression analyses of cells from immunocompetent and immunodeficient mice showed that CD8+ T cells were predominant IFN-gamma producers, and that expansion of CD8+ T cells primed to make IFN-gamma was independent of CD4+ T cells in vivo. Studies in IL-2-deficient mice demonstrated that this cytokine promoted IFN-gamma and IL-4 responses, and ex vivo experiments showed that exogenous IL-2 was required to maintain high-level IFN-gamma production by in vivo-primed CD8+ T cells. Conditions associated with cytokine decreases were accompanied by reduced detectable plasma Ab responses. The results indicate that, although IL-2-dependent CD8+ T cell proliferation does not require endogenous CD4+ T cells, IL-2 production by the CD4+ T cells may promote continued cytokine release from activated CD8+ T cells. By defining these critical steps in cellular and cytokine interactions for shaping endogenous immune responses, the studies advance understanding of the unique conditions regulating CD8+ T cell responses to viral challenges.

Interferon-alpha/beta inhibition of interleukin 12 and interferon-gamma production in vitro and endogenously during viral infection.

Interferon (IFN)-alpha/beta-mediated negative regulation of interleukin 12 (IL-12) and IFN-gamma proteins is reported here. Both IFN-alpha and IFN-beta inhibited fixed Staphylococcus aureus Cowan strain induction of IL-12 and IFN-gamma production by mouse splenic leukocytes in culture. Extended studies with IFN-alpha demonstrated that inhibition was at the level of biologically active IL-12 p70. Effects were selective, as induction of tumor necrosis factor was unaffected and induction of IL-6 was enhanced. Neutralization of IFN-alpha/beta expressed endogenously during infections with murine cytomegalovirus (MCMV) enhanced early IL-12 and IFN-gamma protein production. Furthermore, during infections of mice with lymphocytic choriomeningitis virus (LCMV), this treatment revealed a previously undetected early IL-12 and IFN-gamma protein expression, and mice deficient in IFN-alpha/beta receptor function, but not control mice, also expressed endogenous LCMV-induced IL-12. The effects of IFN-alpha/beta neutralization on production of IL-12 and IFN-gamma during the viral infections were detected in both serum samples and medium conditioned with splenic leukocytes isolated from infected animals. In vitro studies demonstrated that splenic leukocytes isolated from LCMV-infected mice were primed to produce IL-12 in response to stimulation with Staphylococcus aureus Cowan strain, but that this responsiveness was sensitive to added IFN-alpha. Moreover, endogenous IFN-alpha/beta induced by LCMV inhibited in vivo lipopolysaccharide stimulation of IL-12 production. These results demonstrate a new pathway for regulating cytokine responses, and suggest a mechanism for inhibition of IL-12-dependent immune responses during viral infections.

Multiple forms of an inositol polyphosphate 5-phosphatase form signaling complexes with Shc and Grb2.

BACKGROUND: Shc and Grb2 form a complex in cells in response to growth factor stimulation and link tyrosine kinases to Ras during the resulting signaling process. Shc and Grb2 each contain domains that mediate interactions with other unidentified intracellular proteins. For example, the Shc PTB domain binds to 130 kDa and 145 kDa tyrosine-phosphorylated proteins in response to stimulation of cells by growth factors, cytokines and crosslinking of antigen receptors. The Grb2 SH3 domains bind to an unidentified 116 kDa protein in T cells. We have identified three proteins, of 110 kDa, 130 kDa and 145 kDa, as a new family of molecules encoded by the same gene. In vivo studies show that these proteins form signal transduction complexes with Shc and with Grb2. RESULTS: The 130 kDa and 145 kDa tyrosine-phosphorylated proteins that associate with the Shc PTB domain were purified by conventional chromatographic methods. Partial peptide and cDNA sequences corresponding to these proteins, termed SIP-145 and SIP-130 (SIP for signaling inositol polyphosphate 5-phosphatase), identified them as SH2 domain-containing products of a single gene and as members of the inositol polyphosphate 5-phosphatase family. The SIP-130 and SIP-145 proteins and inositol polyphosphate 5-phosphatase activity associated with Shc in vivo in response to B-cell activation. By using an independent approach, expression cloning, we found that the Grb2 SH3 domains bind specifically to SIP-110, a 110 kDa splice variant of SIP-145 and SIP-130, which lacks the SH2 domain. The SIP proteins hydrolyzed phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5)-P3) and Ins (1,3,4,5)-P4, but not PtdIns (4,5)-P2 or Ins (1,4,5)-P3. CONCLUSIONS: These findings strongly implicate the inositol polyphosphate 5-phosphatases in Shc- and Grb2-mediated signal transduction. Furthermore, SIP-110, SIP-130 and SIP-145 prefer 3-phosphorylated substrates, suggesting a link to the phosphatidylinositol 3-kinase signaling pathway.

A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development.

Reduction of food intake and weight gain by the ob protein requires a specific secondary structure and is reversible.

BACKGROUND: Obesity, the condition of excessive accumulation of fat is a poorly understood disorder and is a risk factor for type II diabetes, hypertension, and hyperlipidaemia. Recently, a putative mouse obese gene was cloned and its product, termed ob protein, was shown to be involved in the regulation of body weight. MATERIALS AND METHODS: Bacterial and insect cells were used for expression of recombinant mouse ob protein. Amino-terminal sequence analysis and site-directed mutagenesis were used to identify and characterize the mature form of ob protein. Genetically obese mice and wild-type rats were used to determine the biological activity of ob protein. RESULTS: Mouse ob protein is synthesized as a precursor molecule, the mature form of which was found in mouse serum. Biochemical analysis identified the processing site in the ob precursor molecule and an intramolecular disulfide bond in the mature form that is necessary for activity. Reduction of food intake and weight gain after administration of ob protein to genetically obese mice and wild-type rats is reversible. DISCUSSION: This study demonstrates that ob protein is a secreted satiety factor which regulates body weight and reduces food intake even in animals with no genetic body weight abnormalities. The failure of ob protein to effect these parameters in db/db mice supports the hypothesis that these mice are deficient in a signaling molecule that normally responds to the ob protein.

Endogenous IL-2 contributes to T cell expansion and IFN-gamma production during lymphocytic choriomeningitis virus infection.

IL-2-deficient mice were used to examine the role of endogenous IL-2 for supporting T cell proliferative responses during infection with lymphocytic choriomeningitis virus (LCMV). The studies showed that, although virus-specific CTL activity was induced in the absence of IL-2, the overall magnitude of the response was profoundly inhibited. Examination of proportions and numbers of CD8+ T cells demonstrated that the normal virus-induced expansion of these cells was virtually eliminated in spleens and dramatically decreased in lymph nodes from IL-2-negative mice. Absence of endogenous IL-2 also significantly inhibited virus-induced activated T cell production of IFN-gamma, as well as increases in frequencies and numbers of IFN-gamma-producing cells. Reductions in immune responses were accompanied by impaired viral clearance. Although T cell responses were dramatically reduced in IL-2-deficient, as compared with IL-2-containing mice, activation signals were being delivered in vivo because induced CTLs were sensitive to the cell cycle-specific toxin, hydroxyurea (HU), and CD8+ T cells had induced expression of the IL-2R alpha- and beta-chains. These studies demonstrated that, although low levels of T cell responses can be induced in the absence of IL-2, the factor plays a unique and critical role in supporting T cell proliferative responses in vivo and in optimizing induction of the biologic functions mediated by these cells. Furthermore, the results identify a role for IL-2 in promoting IFN-gamma production in vivo.

Anti-inflammatory properties of a platelet-activating factor acetylhydrolase.

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid that activates cells involved in inflammation. The biological activity of PAF depends on its structural features, namely an ether linkage at the sn-1 position and an acetate group at the sn-2 position. The actions of PAF are abolished by hydrolysis of the acetyl residue, a reaction catalysed by PAF acetylhydrolase. There are at least two forms of this enzyme--one intracellular and another that circulates in plasma and is likely to regulate inflammation. Here we report the molecular cloning and characterization of the human plasma PAF acetylhydrolase. The unique sequence contains a Gly-Xaa-Ser-Xaa-Gly motif commonly found in lipases. Recombinant PAF acetylhydrolase has the substrate specificity and lipoprotein association of the native enzyme, and blocks inflammation in vivo: it markedly decreases vascular leakage in pleurisy and paw oedema, suggesting that PAF acetylhydrolase might be a useful therapy for severe acute inflammation.

A practical cytogenetic protocol for in vitro cytotoxicity and genotoxicity testing.

In vitro cytogenetics has been established as a valid method for evaluating the genotoxic potential of chemical agents. Armstrong et al have described a simple, quantitative approach to in vitro cytotoxicity and genotoxicity testing by using Chinese hamster ovary (CHO) cells. This approach can also be sensitive and repeatable in an inter-laboratory setting, a prerequisite for routine testing of compounds suspected of having genotoxic properties. In the present study, cytotoxicity was evaluated by the parameter of mitotic index (MI). Genotoxicity is measured by the chromosome aberration (Abs) assay as described by Armstrong et al using CHO cells. The basic analytic principles proposed were extended to include human lymphocytes. Sister chromatid exchange (SCE) analysis was used to establish an additional endpoint. Mitomycin C (MMC), an established clastogen, was used as the model compound for protocol validation. Dose response curves for MI and Abs in CHO cells were found to be consistent with those reported by Armstrong et al. Results from our extended study on lymphocytes and using SCE analysis were analogous. Our experience is that this standardized approach is indeed sensitive and reliable and can serve as a basis for an inter-laboratory testing program.

Nerve growth factor in different crystal forms displays structural flexibility and reveals zinc binding sites.

Murine beta-nerve growth factor (beta NGF) is a 118 amino acid residue polypeptide which, as a functional dimer, plays an important role in the survival and development of certain neuronal populations. The structure of the bis-desocta1-8 form of murine beta NGF has been determined in two different crystal modifications using X-ray methods. The two crystal forms, with space groups P2(1)2(1)2(1) and C2, were grown from 18 to 20% polyethylene glycol 8000 and 100 mM Pipes (pH 6.1) with zinc acetate concentrations of 1 mM and 100 mM, respectively. The C2 structure was solved by multiple isomorphous replacement using four heavy-atom derivatives and was refined to a crystallographic residual of 17.9% and 2.5 A resolution. The crystals contain three beta NGF monomers per asymmetric unit. Two monomers form a dimer related by a non-crystallographic 2-fold axis of symmetry. The third monomer also forms a dimer that is very similar, but with a crystallography related monomer as a partner. The electron density clearly defines residues 12 through 115 for all three monomers but the extreme N and C-terminal residues (9 to 11, 116 to 118) are ill defined in some cases. The P2(1)2(1)2(1) structure was solved by molecular replacement using the C2 structure as a search model and was refined to a crystallographic residual of 19.7% at 2.8 A resolution. This crystal form contains two monomers per asymmetric unit, again arranged as a non-crystallographic 2-fold-related dimer. The N and c termini are also variably defined. The core of each of the five monomers, which forms a cysteine knot motif, is very similar in all structures. Also, the dimer structures are very similar to one another, whether the monomers are related by crystallographic or non-crystallographic symmetry. However, three of the four loop regions that extend from the core of each monomer display substantial variability in conformation, even between monomers of the same dimer. This structural variability in the putative receptor binding regions suggests that structural malleability might be important in allowing the ligands to bind to different receptors with different affinities.(ABSTRACT TRUNCATED AT 400 WORDS)

Refinement of the structure of human basic fibroblast growth factor at 1.6 A resolution and analysis of presumed heparin binding sites by selenate substitution.

The three-dimensional structure of human basic fibroblast growth factor has been refined to a crystallographic residual of 16.1% at 1.6 A resolution. The structure has a Kunitz-type fold and is composed of 12 antiparallel beta-strands, 6 of which form a beta-barrel. One bound sulfate ion has been identified in the model, hydrogen bonded to the side chains of Asn 27, Arg 120, and Lys 125. The side chain of Arg 120 has two conformations, both of which permit hydrogen bonds to the sulfate. This sulfate binding site has been suggested as the binding site for heparin (Eriksson, A.E., Cousens, L.S., Weaver, L.H., & Matthews, B.W., 1991, Proc. Natl. Acad. Sci. USA 88, 3441-3445). Two beta-mercaptoethanol (BME) molecules are also included in the model, each forming a disulfide bond to the S gamma atoms of Cys 69 and Cys 92, respectively. The side chain of Cys 92 has two conformations of which only one can bind BME. Therefore the BME molecule is half occupied at this site. The locations of possible sulfate binding sites on the protein were examined by replacing the ammonium sulfate in the crystallization medium with ammonium selenate. Diffraction data were measured to 2.2 A resolution and the structure refined to an R-factor of 13.8%. The binding of the more electron-dense selenate ion was identified at two positions. One position was identical to the sulfate binding site identified previously. The second selenate binding site, which is of lower occupancy, is situated 5.6 A from the first. This ion is hydrogen bonded by the side chain of Lys 135 and Arg 120. Thus the side chain of Arg 120 binds two selenate ions simultaneously. It is suggested that the observed second selenate binding site should also be considered as a possible binding site for heparin, or that both selenate binding sites might simultaneously contribute to the binding of heparin.

Preparation of affinity-fractionated, heparin-derived oligosaccharides and their effects on selected biological activities mediated by basic fibroblast growth factor.

Homogeneously sized, heparin-derived oligosaccharides were prepared from heparin following partial depolymerization with nitrous acid, reduction with sodium borohydride, and fractionation by gel permeation chromatography. The resulting pools of di-, tetra-, hexa-, octa-, and decasaccharides were sequentially applied to an affinity column of human recombinant basic fibroblast growth factor (bFGF) covalently attached to Sepharose 4B and further fractionated into subpools based on their elution from this column in response to gradients of sodium chloride. In general, pools of smaller heparin-derived oligosaccharides required relatively lower salt concentration for complete elution, and pools of larger oligosaccharides required higher salt concentration. The homogeneously sized pools and affinity-fractionated subpools of heparin-derived oligosaccharides were quantitatively assessed as inhibitors or enhancers of specific bFGF-mediated biological activities in five separate assay systems as follows: assay 1, to compete with human lymphoblastoid cells expressing syndecan (RO-12 UC cells) for binding to bFGF-coated wells (Ishihara, M., Tyrrell, D.J., Kiefer, M.C., Barr, P.J., and Swiedler, S.J. (1992) Anal. Biochem. 202, 310-315); assay 2, to inhibit 125I-bFGF binding to "low affinity sites" of adrenocortical endothelial (ACE) cells; assay 3, to inhibit bFGF-induced proliferation of ACE cells; assay 4, to support mitogenic activity of bFGF in a growth stimulation assay of chlorate-treated ACE cells; and assay 5, to enhance the in vitro interaction between 125I-bFGF and the recombinant extra-cellular domain of FGF high affinity receptor. The data derived from the five assay systems demonstrated that heparin-derived hexa- and octasaccharides inhibited the interaction between cell surface heparan sulfate proteoglycan and bFGF (assays 1 and 2) and bFGF-induced proliferation of ACE cells (assay 3) but were unable to enhance the binding of bFGF to its high affinity receptor in vitro (assay 5) or to support bFGF-induced mitogenesis in ACE cells (assay 4). These two activities required at least a decasaccharide with high affinity for bFGF.