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 FGF1⅐HS⅐FGFR1 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 FGF⅐FGFR 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.Heparan sulfate (HS) 1 is a linear and highly sulfated polysaccharide, consisting of 50 -150 basic disaccharide repeats of uronic acid and D-glucosamine units (1). Sulfation can occur at 2-O of the uronic acid and 3-O, 6-O, and N of the D-glucosamine and is catalyzed by a variety of sulfotransferases. Each modification is incomplete, which leads to sequence variation on HS, and it is very likely that critical sulfate groups determine the specificity of HS-protein interactions (2). Along the HS chain, the majority of sulfated residues are clustered in short functional domains separated by relatively less sulfated oligosaccharide sequences (3). Heparin resembles these functional domains and is widely used for the functional study of HS. One major function of HS is to interact with fibroblast growth factors (FGFs) and their receptors (FGFRs) and form FGF⅐HS⅐FGFR signaling complexes (4 -7). Defects in HS can cause complete losses of FGF, Hedgehog, and Wingless signaling pathways and lead to severe abnormality in embryonic development (8, 9). The involvement of HS in the FGF molecular signaling complex suggests that FGF activity and specificity may be modulated by HS and in turn by enzymes that synthesize and degrade HS.FGFs and FGFRs play critical roles in the control of many fundamental cellular processes, such as cell proliferation, differentiation, and migration (10 -13). There are 23 known FGFs and five types of FGFRs in humans (14). FGF1 and FGF2 were the first to be isolated and were called acidic and basic...

PTP is a receptor-type protein-tyrosine phosphatase that is synthesized as a chondroitin sulfate proteoglycan and uses pleiotrophin as a ligand. The chondroitin sulfate portion of this receptor is essential for high affinity binding to pleiotrophin. Here, we purified phosphacan, which corresponds to the extracellular domain of PTP, from postnatal day 7 (P7) and P12 rat cerebral cortex (PG-P7 and PG-P12, respectively) and from P20 rat whole brain (PG-P20). The chondroitin sulfate of these preparations displayed immunologically and compositionally different structures. In particular, only PG-P20 reacted with the monoclonal antibody MO-225, which recognizes chondroitin sulfate containing the GlcA(2S)␤1-3GalNAc(6S) disaccharide unit (D unit). Analysis of the chondroitinase digestion products revealed that GlcA␤1-3GalNAc(4S) disaccharide unit (A unit) was the major component in these preparations and that PG-P20 contained 1.3% D unit, which was not detected in PG-P7 and PG-P12. Interaction analysis using a surface plasmon resonance biosensor indicated that PG-P20 had ϳ5-fold stronger affinity for pleiotrophin (dissociation constant (K D ) ‫؍‬ 0.14 nM) than PG-P7 and PG-P12, although all these preparations showed similar low affinity binding to pleiotrophin after chondroitinase ABC digestion (K D ‫؍‬ 1.4 ϳ 1.6 nM). We also found that shark cartilage chondroitin sulfate D containing ϳ20% D unit bound to pleiotrophin with moderate affinity (K D ‫؍‬ 2.7 nM), whereas whale cartilage chondroitin sulfate A showed no binding to this growth factor. These results suggest that variation of chondroitin sulfate plays important roles in the regulation of signal transduction in the brain.

The CHS2 and CHS3 genes of Candida albicans were disrupted. The double disruptant was still viable. Assessment of chitin and of calcofluor white resistance shows that CHS1 is responsible for septum formation and CHS3 is responsible for overall chitin synthesis otherwise. There were only small differences in virulence to immunocompromised mice of homozygous chs2⌬ and homozygous chs3⌬ null mutants.Like Saccharomyces cerevisiae, Candida albicans harbors three chitin synthase genes, designated CHS1, CHS2, and CHS3 (2, 6, 13). In S. cerevisiae, it was demonstrated by gene disruption experiments that chitin synthase 1 (Chs1p) is involved in the repair of damaged chitin, Chs2p is required for primary septum formation, and Chs3p is responsible for all other chitin syntheses (5,12,14). More recently, Kollar et al. reported that CHS3 also contributes to the formation of linkage between chitin and ␤-1,3-glucan in S. cerevisiae (10). In order to gain more insights into the physiological roles of the chitin synthases of C. albicans, we have disrupted both CHS2 and CHS3 in C. albicans by means of the URA blaster protocol (1).The homozygous chs2⌬ null mutant and the homozygous chs3⌬ null mutant strains of C. albicans were obtained by transforming CAI-4 cells (ura3⌬::imm34/ura3⌬::imm34) with DNA fragments containing either CHS2 in which the hisG-URA3-hisG cassette was inserted at the unique XhoI site or CHS3 in which the 0.8-kb NcoI-ClaI region was replaced by the hisG-URA3-hisG cassette by the lithium acetate method (9). These DNA fragments were successfully integrated into one of the diploid CHS2 or CHS3 alleles, respectively, and the URA3 gene was efficiently eliminated by 5-fluoroorotic acid (5-FOA) selection (11) (Fig. 1). Then these DNA fragments were again transfected into cells in which one of the diploid CHS2 or CHS3 alleles was already flanked by the hisG sequence. Although the second allele of the CHS2 locus was efficiently targeted by the same DNA fragment used to disrupt the first allele, the remaining CHS3 allele was not easily disrupted by transfection of the same DNA fragment. Therefore, we constructed another plasmid in which the hisG-URA3-hisG cassette was inserted at the NcoI site of CHS3. We assumed that use of this DNA for the second round of transfection would increase the efficiency of homologous recombination between the transfected DNA and the remaining intact CHS3 allele because the 0.8-kb NcoI-ClaI region of CHS3 was missing in the already targeted CHS3 locus. As expected, in 3 of 24 uracil auxotrophs, both of the CHS3 alleles were found to be flanked by the hisG sequence after 5-FOA selection, resulting in the homozygous chs3⌬ null mutation (Fig. 1).Cells lacking functional CHS3 grew in a rich medium such as YPD (1% peptone, 2% yeast extract, and 2% dextrose), but their growth was somewhat slower than that of cells missing CHS2 or the parental strain CAI-4 (the doubling times for CAI-4, the homozygous chs2⌬ null mutant, and the homozygous chs3⌬ null mutant were about 70, 72, and 90 min, respectively)...

Saccharomyces cerevisiae GSC1 (also called FKS1) and GSC2 (also called FKS2) have been identified as the genes for putative catalytic subunits of ␤-1,3-glucan synthase. We have cloned three Candida albicans genes, GSC1, GSL1, and GSL2, that have significant sequence homologies with S. cerevisiae GSC1/FKS1, GSC2/FKS2, and the recently identified FKSA of Aspergillus nidulans at both nucleotide and amino acid levels. Like S. cerevisiae Gsc/Fks proteins, none of the predicted products of C. albicans GSC1, GSL1, or GSL2 displayed obvious signal sequences at their N-terminal ends, but each product possessed 10 to 16 potential transmembrane helices with a relatively long cytoplasmic domain in the middle of the protein. Northern blotting demonstrated that C. albicans GSC1 and GSL1 but not GSL2 mRNAs were expressed in the growing yeastphase cells. Three copies of GSC1 were found in the diploid genome of C. albicans CAI4. Although we could not establish the null mutation of C. albicans GSC1, disruption of two of the three GSC1 alleles decreased both GSC1 mRNA and cell wall ␤-glucan levels by about 50%. The purified C. albicans ␤-1,3-glucan synthase was a 210-kDa protein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and all sequences determined with peptides obtained by lysyl endopeptidase digestion of the 210-kDa protein were found in the deduced amino acid sequence of C. albicans Gsc1p. Furthermore, the monoclonal antibody raised against the purified ␤-1,3-glucan synthase specifically reacted with the 210-kDa protein and could immunoprecipitate ␤-1,3-glucan synthase activity. These results demonstrate that C. albicans GSC1 is the gene for a subunit of ␤-1,3-glucan synthase.

Inducible overexpression of the CHS4 gene under the control of the GAL1 promoter increased Chs3p (chitin synthase 3) activity in Saccharomyces cerevisiae several fold. Approximately half of the Chs3p activity in the membranes of cells overexpressing Chs4p was extracted using CHAPS and cholesteryl hemisuccinate. The detergent-extractable Chs3p activity appeared to be non-zymogenic because incubation with trypsin decreased enzyme activity in both the presence and absence of the substrate, UDP-Nacetylglucosamine. Western blotting confirmed that Chs3p was extracted from membranes by CHAPS and cholesteryl hemisuccinate and revealed that Chs4p was also solubilized using these detergents. Yeast two-hybrid analysis with truncated Chs4p demonstrated that the region of Chs4p between amino acids 269 and 563 is indispensable not only for eliciting the non-zymogenic activity of Chs3p but also for binding of Chs4p to Chs3p. Neither the EF-hand motif nor a possible prenylation site in Chs4p was required for these activities. Thus, it was demonstrated that stimulation of non-zymogenic Chs3p activity by Chs4p requires the amino acid region from 269 to 563 of Chs4p, and it seems that Chs4p activates Chs3p through protein-protein interaction.

A search of the yeast data base for a protein homologous to Escherichia coli UDP-N-acetylglucosamine pyrophosphorylase yielded UAP1 (UDP-N-acetylglucosamine pyrophosphorylase), the Saccharomyces cerevisiae gene for UDP-N-acetylglucosamine pyrophosphorylase. The Candida albicans and human homologs were also cloned by screening a C. albicans genomic library and a human testis cDNA library, respectively. Sequence analysis revealed that the human UAP1 cDNA was identical to previously reported AGX1. A null mutation of the S. cerevisiae UAP1 (ScUAP1) gene was lethal, and when expressed under the control of ScUAP1 promoter, both C. albicans and Homo sapiens UAP1 (CaUAP1 and HsUAP1) rescued the ScUAP1-deficient S. cerevisiae cells. All the recombinant ScUap1p, CaUap1p, and HsUap1p possessed UDP-N-acetylglucosamine pyrophosphorylase activities in vitro. The yeast Uap1p utilized N-acetylglucosamine-1-phosphate as the substrate, and together with Agm1p, it produced UDP-N-acetylglucosamine from N-acetylglucosamine-6-phosphate. These results demonstrate that the UAP1 genes indeed specify eukaryotic UDP-GlcNAc pyrophosphorylase and that phosphomutase reaction precedes uridyltransfer. Sequence comparison with other UDP-sugar pyrophosphorylases revealed that amino acid residues, Gly 112

We cloned and characterized a novel Aspergillus nidulans histidine kinase gene, tcsB, encoding a membranetype two-component signaling protein homologous to the yeast osmosensor synthetic lethal N-end rule protein 1 (SLN1), which transmits signals through the high-osmolarity glycerol response 1 (HOG1) mitogen-activated protein kinase (MAPK) cascade in yeast cells in response to environmental osmotic stimuli. From an A. nidulans cDNA library, we isolated a positive clone containing a 3,210-bp open reading frame that encoded a putative protein consisting of 1,070 amino acids. The predicted tcsB protein (TcsB) has two probable transmembrane regions in its N-terminal half and has a high degree of structural similarity to yeast Sln1p, a transmembrane hybrid-type histidine kinase. Overexpression of the tcsB cDNA suppressed the lethality of a temperature-sensitive osmosensing-defective sln1-ts yeast mutant. However, tcsB cDNAs in which the conserved phosphorylation site His 552 residue or the phosphorelay site Asp 989 residue had been replaced failed to complement the sln1-ts mutant. In addition, introduction of the tcsB cDNA into an sln1⌬ sho1⌬ yeast double mutant, which lacked two osmosensors, suppressed lethality in high-salinity media and activated the HOG1 MAPK. These results imply that TcsB functions as an osmosensor histidine kinase. We constructed an A. nidulans strain lacking the tcsB gene (tcsB⌬) and examined its phenotype. However, unexpectedly, the tcsB⌬ strain did not exhibit a detectable phenotype for either hyphal development or morphology on standard or stress media. Our results suggest that A. nidulans has more complex and robust osmoregulatory systems than the yeast SLN1-HOG1 MAPK cascade.Living cells are equipped with mechanisms for sensing environmental stimuli, such as osmotic stress, oxidative stress, and hormones, which allow adaptation to the stimuli through a variety of cellular responses triggered by the sensing and subsequent signaling systems. Two-component signaling systems, which involve a phosphorelay from the histidine of the sensor kinase to the aspartic acid of the response regulator, are widespread in bacteria (23 CaNIK1, and CaSLN1 [19]). Some of the histidine kinases, including yeast SLN1, plant ATHK1, and fungal NIK1 and tcsA, are involved in osmoregulation. In Saccharomyces cerevisiae, Sln1p consists of an extracellular sensor, a kinase, and a response regulator domain in a single polypeptide and is thus a transmembrane hybrid-type histidine kinase (22). Under low-osmolarity conditions, a specific histidine residue within the histidine kinase domain is autophosphorylated. The phosphate moiety of the histidine kinase is transferred to an aspartic acid residue within the response regulator domain and then via a phosphorelay is transferred to the downstream proteins Ypd1p and Ssk1p, shutting off the high-osmolarity glycerol response 1 (HOG1) mitogen-activated protein kinase (MAPK) cascade (24). Histidine kinase activity and phosphorylation of Sln1p are essential for growth at low osm...

Heparan sulfate (HS) binds with various proteins including growth factors, morphogens, and extracellular matrix molecules to regulate their biological functions. These regulatory interactions are considered to be dependent on the structure of HS, which is determined by HS sulfotransferases. To gain insights into the functions of HS sulfotransferases in the development of the nervous system, we examined the expression of these enzymes (3-O-sulfotransferase-1 [3-OST-1], -2, -4; 6-OST-1, -2, -3; and N-deacetylase /N-sulfotransferase-1 [NDST-1], -2, -3) by in situ hybridization and real-time reverse transcription-polymerase chain reaction (RT-PCR). The expression of these genes was spatiotemporally regulated. In the E16 cerebrum, the expression of these genes showed two patterns: (1) selective expression at cortical plate (CP) and ventricular zone (VZ) and (2) wider expression by the cells in the marginal zone (MZ), CP, subplate (SP), and VZ. At P1, most genes showed similar expression patterns, but after P7, these genes were expressed differentially in a layer-specific manner. In the P1 cerebellum, the external granule cell layer (EGL) expressed most genes, the expressions of which were down-regulated at P7. In contrast, Purkinje cells began to express many of these genes after P7. These complex expression patterns suggest that the structure of HS is altered spatiotemporally for regulating various biological activities in the developing brain including the proliferation of neuronal progenitors, extension of axons, and formation of dendrites. We discuss possible functional roles of these sulfotransferases in the signaling of several HS-binding proteins such as fibroblast growth factors, slit, netrin, and sonic hedgehog.