Proceedings: Influence of the oximes HS-3 and HS-6 on soman poisoning in beagles.
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Animals↗
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Immunoprophylaxis against kala-azar: effect of immunization with different doses of EH, and HS strains of L. donovani on challenge with the homologous HS and mouse adopted MS strains.
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Animals↗
The effects of HS-3 and HS-6 on cardiovascular changes in rats caused by soman.
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Animals↗
The spacing of S-domains on HS glycosaminoglycans determines whether the chain is a substrate for intracellular heparanases.
Heparanases are mammalian endoglucuronidases that degrade heparan sulfate (HS) glycosaminoglycans to short 5-6 kDa pieces. In the Golgi, HS glycosaminoglycans are modified by a series of interdependent reactions which result in chains that have regions rich in N- and O-sulfate groups and iduronate residues (S-domains), separated by regions that are nearly devoid of sulfate. Structural analysis of the short HS chains produced by Chinese hamster ovary (CHO) cell heparanases indicate that the enzymes recognize differences in sulfate content between S-domains and unmodified sequences, and cleave the chain at junctions between these regions. To look more closely at whether the spacing of S-domains on the gly- cosaminoglycan influences its ability to be cleaved by heparanases, we examined the susceptibility of the HS chains synthesized by the proteoglycan synthesis mutant, pgsE-606. PGS:E-606 cells are deficient in the modification enzyme N-deacetylase/N-sulfotransferase I, and synthesize HS chains that have fewer N- and O-sulfate groups and iduronate residues compared to wild-type (Bame et al., (1991), J. Biol. Chem., 266, 10287). HS glycosaminoglycans were isolated from wild-type and pgsE-606 cells and separated into populations based on sulfate content. Compared to wild-type HS, which has 14 S-domains, pgsE-606 cells synthesize three HS species, 606-1, 606-2, and 606-3, with 1, 4, and 8 S-domains, respectively. The spacing of the S-domains on the pgsE-606 HS chains is similar to the spacing the modified sequences on wild-type HS, indicating that each mutant glycosaminoglycan is composed of wild-type-like sequences and sequences devoid of S-domains. When incubated with partially purified CHO heparanases, only the portion of the mutant HS chains that had S-domains were degraded. Structural analysis of the heparanase-products confirmed that both the number and the arrangement of S-domains on the HS glycosaminoglycan are important for heparanase susceptibility. The structure of the different pgsE-606 HS chains also suggests mechanisms for the placement of S-domains when the gly- cosaminoglycan is synthesized.
Amidohydrolases↗
Structure and function of the murine beta-globin locus control region 5' HS-3.
We previously identified the murine homologue of the human beta-globin Locus Control Region (LCR) 5' HS-2. The lambda clone containing murine 5' HS-2 extends approximately 12 kb upstream from this site; here, we report the sequence of this entire upstream region. The murine homologue of 5' HS-3 is located approximately 16.0 kb upstream from the mouse epsilon y-globin gene, but no region homologous to human 5' HS-4 was present in our clone. Using a reporter system consisting of a human gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo), we tested murine LCR fragments extending from -21 to -9 kb (with respect to the epsilon y-globin gene cap site) for activity in classical enhancer and integration site assays in K562 and MEL cells. 5' HS-2 behaved as a powerful enhancer and increased the number of productive integration events (as measured by a colony assay) in both K562 and MEL cells. 5' HS-3 had no activity in K562 cells or in transiently transfected MEL cells, but was nearly as active as 5' HS-2 in the MEL cell colony assay. Two additional tests confirmed the identification of murine 5' HS-3: first, a DNA fragment containing 5' HS-3 confers copy number-dependent, integration-site independent inducibility on a linked beta-globin gene in the MEL cell environment. Secondly, a strong DNAseI hypersensitive site maps to the location of the 5' HS-3 functional core in chromatin derived from MEL cells. Collectively, these data suggest that we have identified the murine homologue of human 5' HS-3, and that this site is functional when integrated into the chromatin of MEL cells but not K562 cells. 5' HS-3 may therefore contain information that contributes to the development-specific expression of the beta-like globin genes.
3T3 Cells↗
Alpha 2-HS-glycoprotein: expression in chondrocytes and augmentation of alkaline phosphatase and phospholipase A2 activity.
The alpha 2-HS-glycoprotein is a plasma protein synthesized in liver and enriched in bone. The concentration of alpha 2-HS-glycoprotein dynamically changes in various physiological conditions and is highest in bone during growth, suggesting that it is involved in regulation of endochondral ossification. Northern blot analysis demonstrated that mRNA transcripts from growth zone and resting zone costochondral chondrocyte cultures hybridized with alpha 2-HS-glycoprotein cDNA. However, a difference of mRNA transcript size was observed, with chondrocyte mRNA transcripts being 2.2 kb, while mRNA isolated from liver was 1.6 kb. Presence of alpha 2-HS-glycoprotein in cartilage cells was found by immunohistochemical staining of human fetal epiphyses using anti-human alpha 2-HS-glycoprotein antibody. To understand the role of alpha 2-HS-glycoprotein in cartilage growth, the effects of exogenous alpha 2-HS-glycoprotein were correlated with alkaline phosphatase (ALPase) and phospholipase A2 (PA2) activity in the chondrocyte cultures. Alkaline phosphatase specific activity was stimulated by alpha 2-HS-glycoprotein at concentrations between 0.25 and 1.25 micrograms/mL in the growth zone and resting zone cultures 2.7 and 2.0-fold, respectively. Matrix vesicle PA2 activity was increased only in the growth zone chondrocyte cultures. These results suggested that alpha 2-HS-glycoprotein may contribute to the regulation of the expression of the chondrocyte phenotype. Steady state mRNA levels of ALPase were analyzed in chondrocytes after additions of alpha 2-HS-glycoprotein. The ALPase mRNA levels remained stationary during the stimulation of enzymatic activity, indicating that the effect of alpha 2-HS-glycoprotein upon alkaline phosphatase activity is not at the transcriptional level.
Alkaline Phosphatase↗
Binding of the anti-human sperm monoclonal antibody HS-11 to bull spermatozoa is correlated with fertility in vitro.
The present study aimed to determine if there is bull to bull variation in the binding of the anti-human sperm monoclonal antibody (MAb) HS-11 to bull spermatozoa, and to investigate if there is any correlation between HS-11 binding to spermatozoa and in vitro fertility of the bulls tested. Semen samples of a single collection (split frozen in 0.5-ml straws) from 8 dairy bulls were used. Swim-up separated motile spermatozoa were incubated in 90-microl drops of capacitation medium (TALP+10 microg/ml heparin) at 39 degrees C, 5% CO2, 95% air. At 0, 2, 4 and 6 h of incubation HS-11 was added (1:1000 final concentration), and the MAb binding was assessed by indirect immunofluorescence assay (IIFA). The HS-11 binding was indicated by a bright green fluorescence of the sperm acrosome region. In vitro-matured, good quality bovine oocytes were randomly allocated to spermatozoa of each bull for in vitro fertilization. Sperm samples of 2 to 3 bulls were used in each trial until 4 replicates per bull were attained for IVF (n approximately 100 oocytes/bull) and IIFA experiments. Sperm capacitation status was assessed simultaneously using an egg yolk lysophosphatidylcholine- (LC) induced acrosome reaction assay. The binding of HS-11 to spermatozoa was maximum at 4 h of incubation in most (6/8) of the bull semen samples. Significant (P < 0.01) differences were observed between bulls in the binding of HS-11 to their spermatozoa (range 22 +/- 8 to 52 +/- 5%) at 4 h, but not within replicates. Similarly, variations (P < 0.05) in the cleavage rate were also seen (range 22 +/- 9 to 58 +/- 7%) between bulls. The HS-11 binding and cleavage were significantly correlated (r = 0.43; n = 32; P < 0.05). The highest percentage of spermatozoa underwent acrosome reaction in response to LC treatment at the 4-h incubation period. This and the linear relationship between HS-11 binding and the cleavage rate observed in the present study together strengthen our earlier suggestion that the binding of the monoclonal antibody HS-11 to bull spermatozoa on a time-dependent manner, may indicate capacitation changes. We conclude that 1) between-bull differences exist in HS-11 binding to spermatozoa, and in the cleavage rate, and 2) HS-11 binding to spermatozoa is correlated with fertility, as determined by the cleavage of bovine oocytes matured and fertilized in vitro.
Journal Article↗
Decreased amount of the Rh antigen D in hereditary spherocytosis (HS).
This study was done to determine whether hereditary spherocytosis (HS) red blood cells (RBC) have decreased amounts of Rh antigens. Initially we studied the RBC of five members of one family, two of whom had HS. Using automated quantitative haemagglutination tests, we demonstrated that HS RBC agglutinated less with Rh antisera of four specificities than did normal RBC, indicating that Rh antigens are decreased on HS RBC. In this family, the strength of other blood group antigens on HS RBC was estimated by manual titres and agglutination scores. No appreciable differences in the agglutination of HS and normal RBC were observed with non-Rh antisera. To assess the strength of the D antigen more accurately, the number of D sites was quantitated on the RBC of 19 individuals with HS and 11 of their healthy relatives. HS RBC had 9209 +/- 4084 (mean +/- SD) D sites, whereas the normal RBC had 15 394 +/- 5763 D sites. These two means were significantly different (P less than 0.01). HS RBC were also compared to normal RBC of unrelated individuals who had the same Rh phenotype. These analyses showed that HS RBC had about half of the normal number of D sites. Our data indicate that HS red cells have decreased amount of the Rh antigen D and probably also of other Rh antigens.
Female↗
Transcriptional activation of human zeta 2 globin promoter by the alpha globin regulatory element (HS-40): functional role of specific nuclear factor-DNA complexes.
We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element (HS-40) located 40 kb upstream of the zeta 2 globin gene. It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells, an erythroid cell line of embryonic and/or fetal origin. Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids, they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter. Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104, respectively. The functional domains of HS-40 were also mapped. Bal 31 deletion mapping data suggested that one GATA-1 motif, one GT motif, and two NF-E2/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter. Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor. Finally, we did genomic footprinting of the HS-40 enhancer region in K562 cells, adult nucleated erythroblasts, and different nonerythroid cells. All sequence motifs within the functional core of HS-40, as mapped by transient expression analysis, appeared to bind a nuclear factor(s) in living K562 cells but not in nonerythroid cells. On the other hand, only one of the apparently nonfunctional sequence motifs was bound with factors in vivo. In comparison to K562, nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region. These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer (HS-40) and the globin promoters.
Base Sequence↗
Time-dependent changes of hs-CRP serum concentration in patients with non-ST elevation acute coronary syndrome.
BACKGROUND: Atherosclerosis is typically associated with a low-grade vascular inflammation that can be measured with the highly sensitive C-reactive protein (hs-CRP) assay. Actually, acute coronary syndromes are thought to result from plaque rupture which is induced by the inflammatory process in the atherosclerotic tissue. Therefore, it is interesting to discuss the value of follow-up measurements of hs-CRP in patients with coronary artery disease (CAD). PATIENTS AND METHODS: The hs-CRP concentration of 133 patients consecutively admitted with an acute coronary syndrome (ACS) was measured on admission and after 6 months. The final assessment after 3 years was structured by questionnaire. RESULTS: 17 cardiac events occurred within 6 months, 30 during the following 3 years. The hs-CRP levels (median +/- SEM) decreased significantly (p < 0.001) from baseline (3.9 +/- 1.3 mg/l) to follow-up (2.9 +/- 0.9 mg/l). Subdivision according to cardiac events during the first observation period confirmed this decrease in patients without events (baseline: 3.9 +/- 1.5 mg/l, follow-up: 2.9 +/- 0.9 mg/l; p < 0.001), whereas patients with events showed a persistent elevation (baseline: 3.8 +/- 0.9 mg/l, follow-up: 4.1 +/- 1.0 mg/l; p = 0.426). Patients who developed a further event during the 6-month period showed hs-CRP levels at follow-up that were > 60% of the initial level. Interestingly, 80% of the events within the following 3 years occurred in patients with an hs-CRP level above this discriminator. With a follow-up hs-CRP value above this discriminator the relative risk of suffering an event was 3.4 (95% confidence interval 1.27-8.9; p < 0.05). CONCLUSION: Patients with a non-ST elevation ACS who showed no event within 6 months are characterized by a decrease in hs-CRP levels from baseline to follow-up. Most events in the observation period of 3 years occurred in patients with follow-up hs-CRP levels > 60% of the initial level. Therefore, it was hypothesized that a repeated measurement of hs-CRP levels in CAD patients could help to discriminate those at high risk of further events.
Acute Disease↗
Evaluation of fluorescence hs-CRP immunoassay for point-of-care testing.
BACKGROUND: C-reactive protein (CRP) is one of acute phase respondents that has been used to monitor infection and inflammation episodes. Recent studies have shown that high-sensitivity C-reactive protein (hs-CRP) is a potential risk predictor for future atherosclerosis and cardiovascular diseases (CVD). METHODS: We previously developed a fluorescence-based immunochromatographic method for measuring hs-CRP concentrations (i-CHROMAtrade mark hs-CRP assay) in blood. Whole blood was mixed with detector buffer, and then loaded onto a test cartridge. After 10 min of incubation, the test cartridge was inserted and scanned for acquisition of fluorescence intensity in a laser fluorescence reader (i-CHROMAtrade mark reader). The fluorescence intensity was microprocessed and converted into the concentration of CRP in blood. The test result of 150 samples by the i-CHROMAtrade mark hs-CRP assay method was compared and evaluated with those by TBA 200FR turbidimetry and BN II nephelometry method. The Deming regression and the Bland-Altman difference plot analysis were used for comparison of hs-CRP test result. RESULTS: The i-CHROMAtrade mark hs-CRP assay system exhibited a good linearity with in the whole measuring range (R=0.997). The imprecision of intra- and the inter-assay CVs (coefficient of variation) of assay system were CVs< 3% and < 5% in the range of 0.5-20 mg/l, respectively. The i-CHROMAtrade mark hs-CRP assay method correlated well with TBA 200FR turbidimetry and BN II nephelometry assay method (R=0.988, N=143 and R=0.989, N=143). CONCLUSION: The i-CHROMAtrade mark hs-CRP assay system is comparable to those of other well-known fully automated hs-CRP assay and is suitable for point-of-care testing (POCT) in detection and quantification of hs-CRP.
Adult↗
Posttranslational processing of human alpha 2-HS glycoprotein (human fetuin). Evidence for the production of a phosphorylated single-chain form by hepatoma cells.
alpha 2-HS glycoprotein (alpha 2-HS) is a major protein occurring in human blood and calciferous tissues. Due to extensive sequence identity, alpha 2-HS has been grouped with the fetuins, a family of proteins that occur in fetal plasma in high concentrations. Native alpha 2-HS undergoes a series of posttranslational modifications including proteolytic processing, multiple N-glycosylations and O-glycosylations, and sulfation of the carbohydrate side chains. Various two-chain forms of alpha 2-HS have been prepared from human plasma, however, the single-chain precursor has not yet been isolated. Here, we have studied the biosynthesis of alpha 2-HS by a human hepatoma cell line, HepG2. We demonstrate that a single-chain form and the two-chain form of alpha 2-HS are secreted by this cell line. The alpha 2-HS forms are further modified by phosphorylation on multiple serine residues. Mapping studies indicate that the connecting peptide region releasable from the heavy chain of alpha 2-HS contains at least one such phosphorylation site. Our results identify proteolytic trimming and/or phosphorylation as modifications possibly regulating the biological effects of alpha 2-HS and the homologous fetuins.
Amino Acid Sequence↗
HS-142-1, a potent antagonist of natriuretic peptides in vitro and in vivo.
To determine whether HS-142-1 (HS), a potent atrial natriuretic peptide (ANP) receptor antagonist, also inhibits the effects of brain natriuretic peptide (BNP), urodilatin (URO), and C-type natriuretic peptide, in vitro studies were carried out, demonstrating that HS inhibited production of cGMP by rat fetal lung fibroblast cells induced by ANP, BNP, URO, and C-type natriuretic peptide. Acute clearance studies were conducted in euvolemic Munich-Wistar rats under inactin anesthesia to characterize the effects of HS in vivo. In response to ANP, BNP, or URO (4 micrograms/kg priming dose plus 0.5 micrograms/kg per minute for 20 min), urine flow, absolute sodium excretion rates, and fractional sodium excretion exhibited similar increases (four- to fivefold) in vehicle-treated rats; these responses were, however, completely abolished by prior HS treatment. The tendency for GFR to rise during the infusion of natriuretic peptides (NP) was also blocked after HS. By contrast, HS did not block the renal effects of L-arginine, a precursor of nitric oxide, or of furosemide. Furthermore, the inhibition of endogenous NP by HS was associated with small but significant reductions in GFR and absolute and fractional sodium excretion in normal rats under euvolemic but not hydropenic conditions. These studies provide evidence that the observed effects of HS in vivo and in vitro are mediated exclusively by receptors of NP. Together, these data support the view that HS is a highly specific ligand for NP receptors, capable of antagonizing the renal effects not only of exogenous ANP, but also those of BNP and URO.
Animals↗
Localization and inhibitory activity of alpha(2)HS-glycoprotein in the kidney.
alpha(2)HS-Glycoprotein (HS), a crystal surface binding substance extracted from human urine, is considered to be one of the urinary macromolecular inhibitors in urolithiasis. In the present study, reverse transcription-polymerase chain reaction was used to examine HS mRNA expression, and immunohistochemical staining was used to reveal its localization in the human kidney. The inhibitory effects of recombinant human HS and native human HS on calcium oxalate crystal growth were examined in a seed crystal system. HS mRNA was found to be expressed in the human kidney, and it was located in the epithelial cells of distal and proximal renal tubular cells. However, neither recombinant HS nor native HS had an inhibitory effect on crystals in the protein concentration of urine of healthy humans. HS in the urine, therefore, does not seem to be a potent inhibitor in stone formation.
Blood Proteins↗
Production and characterization of monoclonal and polyclonal antibodies to human alpha 2-HS: development of a two-site ELISA test.
A convenient and sensitive indirect sandwich ELISA test was developed for measuring both 63 kDa human alpha 2-HS secreted by human hepatoma cell lines and the 59 kDa alpha 2-HS species present in serum/plasma. Monoclonal and rabbit antibodies to plasma alpha 2-HS were produced and selected by immunoprecipitation techniques using iodinated alpha 2-HS or 35S-labeled alpha 2-HS. Various monoclonal antibodies recognizing both forms of the protein were coated onto microtiter plates and after binding of alpha 2-HS, biotinylated monoclonal antibodies with compatible binding or biotinylated immunopurified F(ab')2 fragments from the rabbit antiserum were added and subsequently revealed with avidin-biotin peroxidase complex. Formats using a rabbit detector antibody were the most sensitive and one was selected for the whole study. The test developed was capable of detecting plasma alpha 2-HS devoid of connecting peptide and HepG2 hepatoma cell line derived alpha 2-HS at the ng/ml level. The test has been used to measure levels of alpha 2-HS in both serum and supernatants from HepG2 cell lines.
Animals↗
The arrangement of disulfide loops in human alpha 2-HS glycoprotein. Similarity to the disulfide bridge structures of cystatins and kininogens.
The complete disulfide loop structure of human alpha 2-HS glycoprotein has been elucidated. alpha 2-HS glycoprotein isolated from human plasma was found to be a two-chain protein composed of a heavy and a light chain. The heavy chain comprises the A-chain of alpha 2-HS glycoprotein (Yoshioka, Y., Gejyo, F., Marti, T., Rickli, E. E., Bürgi, W., Offner, G. D., Troxler, R. F., and Schmid, K. (1986) J. Biol. Chem. 261, 1665-1676) and part of the connecting peptide which has been predicted from the corresponding cDNA sequence (Lee, C. C., Bowman, B. H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4403-4407), whereas the light chain corresponds to the beta-chain of alpha 2-HS glycoprotein (Gejyo, F., Chang, J. L., Bürgi, W., Schmid, K., Offner, G. D., Troxler, R. F., Van Halbeek, H., Dorland, L., Gerwig, G. J., Vliegenthart, J. F. G. (1983) J. Biol. Chem. 258, 4966-4971). Twelve half-cystine residues are present in the alpha 2-HS glycoprotein molecule, and 11 of them are positioned in the heavy chain and a single one in the light chain of the molecule; they form six disulfide bridges. The first and the last half-cystine residues of the amino acid sequence of alpha 2-HS glycoprotein are engaged in the formation of a loop spanning the extreme NH2- and COOH-terminal portions of the molecule, thereby connecting the heavy and light chains. The other 10 half-cystines residues are linked consecutively in the heavy chain and form five loops which span 4-19 amino acid residues. Among them are two pairs of loops which are characterized by mutual sequence homology. The particular arrangement of disulfide loops in alpha 2-HS glycoprotein is similar to the patterns of linearly arranged and tandemly repeated disulfide loops of cysteine proteinase inhibitors, i.e. the cystatins and the kininogens. It is concluded that alpha 2-HS glycoprotein represents a structural prototype of a novel family among the cystatin superfamily, characterized by the presence of two cystatin-like building blocks. Extensive similarity among the NH2-terminal sequences of alpha 2-HS glycoprotein and human histidine-rich glycoprotein suggest that the latter protein is another candidate protein of this new family.
Amino Acid Sequence↗
Acute myeloid leukemia cells are protected from spontaneous and drug-induced apoptosis by direct contact with a human bone marrow stromal cell line (HS-5).
In vitro culture systems that parallel in vivo growth conditions are needed to study leukemia biology and to accurately test therapeutic efficacies. We investigated the effects of the HS-5 human bone marrow stromal cell line on cultured primary leukemia cell survival and chemosensitivity.A total of 30 bone marrow (BM) samples from untreated acute myeloid leukemia (AML) patients were cultured for 96 hours with serum and growth factors, without HS-5, or in direct contact with HS-5 monolayers, or with HS-5 separated from AML cells by transwell inserts. In some experiments, cytosine arabinoside or daunomycin was added for the last 18 hours of culture. Apoptosis frequencies, bcl-2 protein expression, proliferating cell nuclear antigen expression, and cell cycle distributions were determined in four-color flow cytometry analyses of CD45(+) leukemia cells. In comparison to control growth conditions, direct contact with HS-5 significantly inhibited culture-induced and drug-induced apoptosis of AML cells. Direct contact of AML cells with HS-5 significantly increased short-term proliferation and viability, and colony formation of primary AML cells. HS-5-mediated apoptosis inhibition was not consistently associated with increased bcl-2 protein in AML cells. Noncontact conditions inhibited drug-induced apoptosis significantly less than direct contact with HS-5. Coculture of AML cells on HS-5 monolayers improved in vitro leukemia cell survival and attenuated chemotherapy-induced leukemia cell killing. This has practical significance, increasing the fraction of primary AML samples that can be analyzed in vitro, and allows drug sensitivity testing in growth conditions more similar to the bone marrow microenvironment.
Antineoplastic Agents↗
The involvement of heparan sulfate (HS) in FGF1/HS/FGFR1 signaling complex.
Fibroblast growth factor (FGF) signaling begins with the formation of a ternary complex of FGF, FGF receptor (FGFR), and heparan sulfate (HS). Multiple models have been proposed for the ternary complex. However, major discrepancies exist among those models, and none of these models have evaluated the functional importance of the interacting regions on the HS chains. To resolve the discrepancies, we measured the size and molar ratio of HS in the complex and showed that both FGF1 and FGFR1 simultaneously interact with HS; therefore, a model of 2:2:2 FGF1.HS.FGFR1 was shown to fit the data. Using genetic and biochemical methods, we generated HSs that were defective in FGF1 and/or FGFR1 binding but could form the signaling ternary complex. Both genetically and chemically modified HSs were subsequently assessed in a BaF3 cell mitogenic activity assay. The ability of HS to support the ternary complex formation was found to be required for FGF1-stimulated cell proliferation. Our data also proved that specific critical groups and sites on HS support complex formation. Furthermore, the molar ratio of HS, FGF1, and FGFR1 in the ternary complex was found to be independent of the size of HS, which indicates that the selected model can take place on the cell surface proteoglycans. Finally, a mechanism for the FGF.FGFR signaling complex formation on cell membrane was proposed, where FGF and FGFR have their own binding sites on HS and a distinct ternary complex formation site is directly responsible for mitogenic activity.
Cell Line↗