Structure of the glycosylphosphatidylinositol anchor of an arabinogalactan protein from Pyrus communis suspension-cultured cells

Arabinogalactan proteins (AGPs) are proteoglycans of higher plants, which are implicated in growth and development. We recently have shown that two AGPs, NaAGP1 (from Nicotiana alata styles) and PcAGP1 (from Pyrus communis cell suspension culture), are modified by the addition of a glycosylphosphatidylinositol (GPI) anchor. However, paradoxically, both AGPs were buffer soluble rather than membrane associated. We now show that pear suspension cultured cells also contain membrane-bound GPI-anchored AGPs. This GPI anchor has the minimal core oligosaccharide structure, d-Manα(1–2)-d-Manα(1–6)-d-Manα(1–4)-d-GlcN-inositol, which is consistent with those found in animals, protozoa, and yeast, but with a partial β(1–4)-galactosyl substitution of the 6-linked Man residue, and has a phosphoceramide lipid composed primarily of phytosphingosine and tetracosanoic acid. The secreted form of PcAGP1 contains a truncated GPI lacking the phosphoceramide moiety, suggesting that it is released from the membrane by the action of a phospholipase D. The implications of these findings are discussed in relation to the potential mechanisms by which GPI-anchored AGPs may be involved in signal transduction pathways.

STEAP: A prostate-specific cell-surface antigen highly expressed in human prostate tumors

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell–cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging.

Attachment to the nuclear matrix mediates specific alterations in chromatin structure

The DNA in eukaryotic chromosomes is organized into a series of loops that are permanently attached at their bases to the nuclear scaffold or matrix at sequences known as scaffold-attachment or matrix-attachment regions. At present, it is not clear what effect affixation to the nuclear matrix has on chromatin architecture in important regulatory regions such as origins of replication or the promoter regions of genes. In the present study, we have investigated cell-cycle-dependent changes in the chromatin structure of a well characterized replication initiation zone in the amplified dihydrofolate reductase domain of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Replication can initiate at any of multiple potential sites scattered throughout the 55-kilobase intergenic region in this domain, with two subregions (termed ori-β and ori-γ) being somewhat preferred. We show here that the chromatin in the ori-β and ori-γ regions undergoes dramatic alterations in micrococcal nuclease hypersensitivity as cells cross the G1/S boundary, but only in those copies of the amplicon that are affixed to the nuclear matrix. In contrast, the fine structure of chromatin in the promoter of the dihydrofolate reductase gene does not change detectably as a function of matrix attachment or cell-cycle position. We suggest that attachment of DNA to the nuclear matrix plays an important role in modulating chromatin architecture, and this could facilitate the activity of origins of replication.

Transgenic knockout mice exclusively expressing human hemoglobin S after transfer of a 240-kb βs-globin yeast artificial chromosome: A mouse model of sickle cell anemia

Sickle cell anemia (SCA) and thalassemia are among the most common genetic diseases worldwide. Current approaches to the development of murine models of SCA involve the elimination of functional murine α- and β-globin genes and substitution with human α and βs transgenes. Recently, two groups have produced mice that exclusively express human HbS. The transgenic lines used in these studies were produced by coinjection of human α-, γ-, and β-globin constructs. Thus, all of the transgenes are integrated at a single chromosomal site. Studies in transgenic mice have demonstrated that the normal gene order and spatial organization of the members of the human β-globin gene family are required for appropriate developmental and stage-restricted expression of the genes. As the cis-acting sequences that participate in activation and silencing of the γ- and β-globin genes are not fully defined, murine models that preserve the normal structure of the locus are likely to have significant advantages for validating future therapies for SCA. To produce a model of SCA that recapitulates not only the phenotype, but also the genotype of patients with SCA, we have generated mice that exclusively express HbS after transfer of a 240-kb βs yeast artificial chromosome. These mice have hemolytic anemia, 10% irreversibly sickled cells in their peripheral blood, reticulocytosis, and other phenotypic features of SCA.

The α1 domain of HLA-G1 and HLA-G2 inhibits cytotoxicity induced by natural killer cells: Is HLA-G the public ligand for natural killer cell inhibitory receptors?

We have investigated the protective role of the membrane-bound HLA-G1 and HLA-G2 isoforms against natural killer (NK) cell cytotoxicity. For this purpose, HLA-G1 and HLA-G2 cDNAs were transfected into the HLA class I-negative human K562 cell line, a known reference target for NK lysis. The HLA-G1 protein, encoded by a full-length mRNA, presents a structure similar to that of classical HLA class I antigens. The HLA-G2 protein, deduced from an alternatively spliced transcript, consists of the α1 domain linked to the α3 domain. In this study we demonstrate that (i) HLA-G2 is present at the cell surface as a truncated class I molecule associated with β2-microglobulin; (ii) NK cytolysis, observed in peripheral blood mononuclear cells and in polyclonal CD3− CD16+ CD56+ NK cells obtained from 20 donors, is inhibited by both HLA-G1 and HLA-G2; this HLA-G-mediated inhibition is reversed by blocking HLA-G with a specific mAb; this led us to the conjecture that HLA-G is the public ligand for NK inhibitory receptors (NKIR) present in all individuals; (iii) the α1 domain common to HLA-G1 and HLA-G2 could mediate this protection from NK lysis; and (iv) when transfected into the K562 cell line, both HLA-G1 and HLA-G2 abolish lysis by the T cell leukemia NK-like YT2C2 clone due to interaction between the HLA-G isoform on the target cell surface and a membrane receptor on YT2C2. Because NKIR1 and NKIR2, known to interact with HLA-G, were undetectable on YT2C2, we conclude that a yet-unknown specific receptor for HLA-G1 and HLA-G2 is present on these cells.

The structure and precision of retinal spike trains

Assessing the reliability of neuronal spike trains is fundamental to an understanding of the neural code. We measured the reproducibility of retinal responses to repeated visual stimuli. In both tiger salamander and rabbit, the retinal ganglion cells responded to random flicker with discrete, brief periods of firing. For any given cell, these firing events covered only a small fraction of the total stimulus time, often less than 5%. Firing events were very reproducible from trial to trial: the timing jitter of individual spikes was as low as 1 msec, and the standard deviation in spike count was often less than 0.5 spikes. Comparing the precision of spike timing to that of the spike count showed that the timing of a firing event conveyed several times more visual information than its spike count. This sparseness and precision were general characteristics of ganglion cell responses, maintained over the broad ensemble of stimulus waveforms produced by random flicker, and over a range of contrasts. Thus, the responses of retinal ganglion cells are not properly described by a firing probability that varies continuously with the stimulus. Instead, these neurons elicit discrete firing events that may be the fundamental coding symbols in retinal spike trains.

Genomic structure and ecdysone regulation of the prophenoloxidase 1 gene in the malaria vector Anopheles gambiae

Prophenoloxidase, a melanin-synthesizing enzyme, is considered to be an important arthropod immune protein. In mosquitoes, prophenoloxidase has been shown to be involved in refractory mechanisms against malaria parasites. In our study we used Anopheles gambiae, the most important human malaria vector, to characterize the first arthropod prophenoloxidase gene at the genomic level. The complete nucleotide sequence, including the immediate 5′ flanking sequence (−855 bp) of the prophenoloxidase 1 gene, was determined. The gene spans 10 kb and is composed of five exons and four introns coding for a 2.5-kb mRNA. In the 5′ flanking sequence, we found several putative regulatory motifs, two of which were identified as ecdysteroid regulatory elements. Electrophoretic mobility gel-shift assays and supershift assays demonstrated that the Aedes aegypti ecdysone receptor/Ultraspiracle nuclear receptor complex, and, seemingly, the endogenous Anopheles gambiae nuclear receptor complex, was able to bind one of the ecdysteroid response elements. Furthermore, 20-hydroxyecdysone stimulation was shown to up-regulate the transcription of the prophenoloxidase 1 gene in an A. gambiae cell line.

Atomic structure of a thermostable subdomain of HIV-1 gp41

Infection by HIV-1 involves the fusion of viral and cellular membranes with subsequent transfer of viral genetic material into the cell. The HIV-1 envelope glycoprotein that mediates fusion consists of the surface subunit gp120 and the transmembrane subunit gp41. gp120 directs virion attachment to the cell–surface receptors, and gp41 then promotes viral–cell membrane fusion. A soluble, α-helical, trimeric complex within gp41 composed of N-terminal and C-terminal extraviral segments has been proposed to represent the core of the fusion-active conformation of the HIV-1 envelope. A thermostable subdomain denoted N34(L6)C28 can be formed by the N-34 and C-28 peptides connected by a flexible linker in place of the disulfide-bonded loop region. Three-dimensional structure of N34(L6)C28 reveals that three molecules fold into a six-stranded helical bundle. Three N-terminal helices within the bundle form a central, parallel, trimeric coiled coil, whereas three C-terminal helices pack in the reverse direction into three hydrophobic grooves on the surface of the N-terminal trimer. This thermostable subdomain displays the salient features of the core structure of the isolated gp41 subunit and thus provides a possible target for therapeutics designed selectively to block HIV-1 entry.

Subunit structure of the mammalian exocyst complex

The exocyst is a protein complex required for the late stages of secretion in yeast. Unlike the SNAREs (SNAP receptors), important secretory proteins that are broadly distributed on the target membrane, the exocyst is specifically located at sites of vesicle fusion. We have isolated cDNAs encoding the rexo70, rsec5, and rsec15 subunits of the mammalian complex. The amino acid sequences encoded by these genes are between 21% and 24% identical to their yeast homologs. All three genes are broadly expressed and multiple transcripts are observed for rexo70 and rsec15. Characterization of cDNAs encoding the 84-kDa subunit of the mammalian complex revealed a novel protein. mAbs were generated to the mammalian rsec6 subunit of the exocyst complex. rsec6 immunoreactivity is found in a punctate distribution at terminals of PC12 cell processes at or near sites of granule exocytosis.

Interaction of Fibroblast Growth Factor-2 (FGF-2) with Free Gangliosides: Biochemical Characterization and Biological Consequences in Endothelial Cell Cultures

Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated. Here, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated. Size exclusion chromatography demonstrates that native, but not heat-denatured, 125I-FGF-2 binds to micelles formed by gangliosides GT1b, GD1b, or GM1. Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT1b > GD1b > GM1 = GM2 = sulfatide > GM3 = galactosyl-ceramide, whereas asialo-GM1, neuraminic acid, and N-acetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM1 to immobilized FGF-2 indicates that FGF–2/GM1 interaction occurs with a Kd equal to 6 μM. This interaction is inhibited by the sialic acid-binding peptide mastoparan and by the synthetic fragments FGF-2(112–129) and, to a lesser extent, FGF-2(130–155), whereas peptides FGF-2(10–33), FGF-2(39–59), FGF-2(86–96), and the basic peptide HIV-1 Tat(41–60) were ineffective. These data identify the COOH terminus of FGF-2 as a putative ganglioside-binding region. Exogenous gangliosides inhibit the binding of 125I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT1b > GD1b > GM1 > sulfatide a = sialo-GM1. Accordingly, GT1b,GD1b, GM1, and GM2, but not GM3 and asialo-GM1, prevent the binding of 125I-FGF-2 to a soluble, recombinant form of extracellular FGFR-1. Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM1 to immobilized FGF-2. In agreement with their FGFR antagonist activity, free gangliosides inhibit the mitogenic activity exerted by FGF-2 on endothelial cells in the same range of concentrations. Also in this case, GT1b was the most effective among the gangliosides tested while asialo-GM1, neuraminic acid, N-acetylneuramin-lactose, galactosyl-ceramide, and sulfatide were ineffective. In conclusion, the data demonstrate the capacity of exogenous gangliosides to interact with FGF-2. This interaction involves the COOH terminus of the FGF-2 molecule and depends on the structure of the oligosaccharide chain and on the presence of sialic acid residue(s) in the ganglioside molecule. Exogenous gangliosides act as FGF-2 antagonists when added to endothelial cell cultures. Since gangliosides are extensively shed by tumor cells and reach elevated levels in the serum of tumor-bearing patients, our data suggest that exogenous gangliosides may affect endothelial cell function by a direct interaction with FGF-2, thus modulating tumor neovascularization.

The Cell Surface Flocculin Flo11 Is Required for Pseudohyphae Formation and Invasion by Saccharomyces cerevisiae

Diploid yeast develop pseudohyphae in response to nitrogen starvation, while haploid yeast produce invasive filaments which penetrate the agar in rich medium. We have identified a gene, FLO11, that encodes a cell wall protein which is critically required for both invasion and pseudohyphae formation in response to nitrogen starvation. FLO11 encodes a cell surface flocculin with a structure similar to the class of yeast serine/threonine-rich GPI-anchored cell wall proteins. Cells of the Saccharomyces cerevisiae strain Σ1278b with deletions of FLO11 do not form pseudohyphae as diploids nor invade agar as haploids. In rich media, FLO11 is regulated by mating type; it is expressed in haploid cells but not in diploids. Upon transfer to nitrogen starvation media, however, FLO11 transcripts accumulate in diploid cells, but not in haploids. Overexpression of FLO11 in diploid cells, which are otherwise not invasive, enables them to invade agar. Thus, the mating type repression of FLO11 in diploids grown in rich media suffices to explain the inability of these cells to invade. The promoter of FLO11 contains a consensus binding sequence for Ste12p and Tec1p, proteins known to cooperatively activate transcription of Ty1 elements and the TEC1 gene during development of pseudohyphae. Yeast with a deletion of STE12 does not express FLO11 transcripts, indicating that STE12 is required for FLO11 expression. These ste12-deletion strains also do not invade agar. However, the ability to invade can be restored by overexpressing FLO11. Activation of FLO11 may thus be the primary means by which Ste12p and Tec1p cause invasive growth.

ARF Is Required for Maintenance of Yeast Golgi and Endosome Structure and Function

ADP ribosylation factor (ARF) is thought to play a critical role in recruiting coatomer (COPI) to Golgi membranes to drive transport vesicle budding. Yeast strains harboring mutant COPI proteins exhibit defects in retrograde Golgi to endoplasmic reticulum protein transport and striking cargo-selective defects in anterograde endoplasmic reticulum to Golgi protein transport. To determine whether arf mutants exhibit similar phenotypes, the anterograde transport kinetics of multiple cargo proteins were examined in arf mutant cells, and, surprisingly, both COPI-dependent and COPI-independent cargo proteins exhibited comparable defects. Retrograde dilysine-mediated transport also appeared to be inefficient in the arf mutants, and coatomer mutants with no detectable anterograde transport defect exhibited a synthetic growth defect when combined with arf1Δ, supporting a role for ARF in retrograde transport. Remarkably, we found that early and medial Golgi glycosyltransferases localized to abnormally large ring-shaped structures. The endocytic marker FM4–64 also stained similar, but generally larger ring-shaped structures en route from the plasma membrane to the vacuole in arf mutants. Brefeldin A similarly perturbed endosome morphology and also inhibited transport of FM4–64 from endosomal structures to the vacuole. Electron microscopy of arf mutant cells revealed the presence of what appear to be hollow spheres of interconnected membrane tubules which likely correspond to the fluorescent ring structures. Together, these observations indicate that organelle morphology is significantly more affected than transport in the arf mutants, suggesting a fundamental role for ARF in regulating membrane dynamics. Possible mechanisms for producing this dramatic morphological change in intracellular organelles and its relation to the function of ARF in coat assembly are discussed.

Effects of Genome Position and the DNA Damage Checkpoint on the Structure and Frequency of sod2 Gene Amplification in Fission Yeast

The Schizosaccharomyces pombe sod2 gene, located near the telomere on the long arm of chromosome I, encodes a Na+ (or Li+)/H+ antiporter. Amplification of sod2 has previously been shown to confer resistance to LiCl. We analyzed 20 independent LiCl-resistant strains and found that the only observed mechanism of resistance is amplification of sod2. The amplicons are linear, extrachromosomal elements either 225 or 180 kb long, containing both sod2 and telomere sequences. To determine whether proximity to a telomere is necessary for sod2 amplification, a strain was constructed in which the gene was moved to the middle of the same chromosomal arm. Selection of LiCl-resistant strains in this genetic background also yielded amplifications of sod2, but in this case the amplified DNA was exclusively chromosomal. Thus, proximity to a telomere is not a prerequisite for gene amplification in S. pombe but does affect the mechanism. Relative to wild-type cells, mutants with defects in the DNA damage aspect of the rad checkpoint control pathway had an increased frequency of sod2 amplification, whereas mutants defective in the S-phase completion checkpoint did not. Two models for generating the amplified DNA are presented.

The Role of the Schizosaccharomyces pombe gar2 Protein in Nucleolar Structure and Function Depends on the Concerted Action of its Highly Charged N Terminus and its RNA-binding Domains

Nonribosomal nucleolar protein gar2 is required for 18S rRNA and 40S ribosomal subunit production in Schizosaccharomyces pombe. We have investigated the consequences of the absence of each structural domain of gar2 on cell growth, 18S rRNA production, and nucleolar structure. Deletion of gar2 RNA-binding domains (RBDs) causes stronger inhibition of growth and 18S rRNA accumulation than the absence of the whole protein, suggesting that other factors may be titrated by its remaining N-terminal basic/acidic serine-rich domain. These drastic functional defects correlate with striking nucleolar hypertrophy. Point mutations in the conserved RNP1 motifs of gar2 RBDs supposed to inhibit RNA–protein interactions are sufficient to induce severe nucleolar modifications but only in the presence of the N-terminal domain of the protein. Gar2 and its mutants also distribute differently in glycerol gradients: gar2 lacking its RBDs is found either free or assembled into significantly larger complexes than the wild-type protein. We propose that gar2 helps the assembly on rRNA of factors necessary for 40S subunit synthesis by providing a physical link between them. These factors may be recruited by the N-terminal domain of gar2 and may not be released if interaction of gar2 with rRNA is impaired.

Mutational Effect of Fission Yeast Polα on Cell Cycle Events

Polα is the principal DNA polymerase for initiation of DNA replication and also functions in postinitiation DNA synthesis. In this study, we investigated the cell cycle responses induced by mutations in polα+. Germinating spores carrying either a deletion of polα+ (polαΔ) or a structurally intact but catalytically dead polα mutation proceed to inappropriate mitosis with no DNA synthesis. This suggests that the catalytic function, and not the physical presence of Polα, is required to generate the signal that prevents the cells from entering mitosis prematurely. Cells with a polαts allele arrest the cell cycle near the hydroxyurea arrest point, but, surprisingly, polαts in cdc20 (polε mutant) background arrested with a cdc phenoytpe, not a polαts-like phenotype. At 25°C, replication perturbation caused by polαts alleles induces Cds1 kinase activity and requires the checkpoint Rads, Cds1, and Rqh1, but not Chk1, to maintain cell viability. At 36°C, replication disruption caused by polαts alleles induces the phosphorylation of Chk1; however, mutant cells arrest with heterogeneous cell sizes with a population of the cells entering aberrant mitosis. Together, our results indicate that the initiation DNA structure synthesized by Polα is required to bring about the S phase to mitosis checkpoint, whereas replication defects of different severity caused by polαts mutations induce differential downstream kinase responses.