Partial characterization of the N-linked oligosaccharide structures on P-selectin glycoprotein ligand-1 (PSGL-1).

PSGL-1, a specific ligand for P-, E- and L-selectin, was isolated from in vivo [3H]-glucosamine labeled HL-60 cells by a combination of wheat germ agglutinin-agarose and P- or E-selectin-agarose chromatography. N-linked oligosaccharides were released from the purified, denatured ligand molecule by peptide: N-glycosidase F treatment and, following separation by Sephacryl S-200 chromatography, partially characterized using lectin, ion-exchange and size-exclusion chromatography in combination with glycosidase digestions. The data obtained suggest that the N-glycans on PSGL-1 are predominantly core-fucosylated, multiantennary complex type structures with extended, poly-N-acetyllactosamine containing outer chains. A portion of the outer chains appears to be substituted with fucose indicating that the N-glycans, in addition to the O-glycans on PSGL-1, may be involved in selectin binding.

Characterization of the O-linked oligosaccharide structures on P-selectin glycoprotein ligand-1 (PSGL-1).

P-selectin glycoprotein ligand-1, PSGL-1, a specific ligand for P-, E-, and L-selectin, was isolated from in vivo [3H]-glucosamine labeled HL-60 cells by a combination of wheat germ agglutinin and platelet P-selectin- or E-selectin receptor globulin-agarose chromatography. The O-linked oligosaccharides on the ligand were released by mild alkaline sodium borohydride treatment and analyzed by a combination of ion-exchange, size exclusion, lectin, and paper chromatography, together with specific exoglycosidase treatments and chemical modifications. Approximately 91% of the radioactivity released from PSGL-1 was recovered in five O-linked glycans: GalNAc (approximately 4% of the total structures), Galp, 3GalNAc (36%), and Galbeta, 3GalNAc substituted with one (45%), two (6%), or three (3%) N-acetyllactosamine repeat units. None of these structures contained fucose, and the majority were substituted with at least one sialic acid. The N-acetyllactosmine-containing structures appeared to be core 2. The remaining 9% of the radioactivity recovered in O-linked oligosaccharides from PSGL-1, eluted in two peaks at 11.8 and 10.2 glucose units, on size-exclusion chromatography. Results from lectin chromatography and chemical and enzymatic degradation experiments suggest that the major portion of the radioactivity in these peaks is associated with sialylated N-acetyllactosamine-type oligosaccharides, substituted with fucose at the penultimate residue in the nonreducing end. Since both sialic acid and fucose reportedly are crucial requirements for selectin binding, these results suggest that only a minor portion, approximately 4.5%, of the O-linked oligosaccharides on PSGL-1 are involved in the interaction with the selectins.

The P-selectin glycoprotein ligand functions as a common human leukocyte ligand for P- and E-selectins.

P- and E-selectins belong to a family of Ca(2+)-dependent lectins and function as receptors for myeloid leukocytes. We have described a panel of monoclonal antibodies which recognize a sialoglycoprotein from human neutrophils and HL-60 promyelocytic cells and inhibit adhesion of these cells to P-selectin. In this study, we show that the E-selectin receptor-globulin (E-selectin Rg) affinity chromatography can isolate specifically only one glycoprotein from [3H]glucosamine-labeled HL-60 cells in a Ca(2+)-dependent manner. This protein has a molecular mass of approximately 120 kDa under reducing conditions, which appears to be identical with the previously characterized glycoprotein ligand for P-selectin. The molecule can be cross-depleted by and cross-bound to the E- and P-selectin columns. The chromatographic profile of desialylated O-linked carbohydrates from molecules purified by P- and E-selectin affinity chromatography are identical. Both have five structures at 12.8, 9.8, 6.3, 3.5, and 2.5 glucose units. PL5 monoclonal antibody to the P-selectin sialoglycoprotein ligand, E-selectin Rg, and antiserum to P-selectin glycoprotein ligand-1 (PSGL-1) all recognize the purified P-selectin ligand on ligand blots and immunoblots. Furthermore, PL5 monoclonal antibody blocks adhesion of HL-60 cells and human neutrophils to E-selectin Rg. Taken together, our results demonstrate that the P- and E-selectin ligand defined in this study is PSGL-1 and suggest that this molecule is an important leukocyte ligand for both P- and E-selectins.

A sialoglycoprotein from human leukocytes functions as a ligand for P-selectin.

P-selectin (CD62P), a Ca(2+)-dependent lectin expressed on activated platelets and endothelial cells, functions as a receptor for myeloid and monocytoid cells. Previous reports have described a homodimeric sialoglycoprotein from human leukocytes and HL-60 cells specifically recognized by P-selectin. We describe here a panel of monoclonal antibodies prepared against high molecular weight fractions of HL-60 cell membranes. These antibodies are of IgM isotype, bind to a approximately 240-kDa protein from human leukocyte membranes which is also reactive with P-selectin. They recognize a Ca(2+)-dependent, sialidase-sensitive determinant on myeloid and monocytoid cell lines. Each antibody specifically inhibits adhesion of neutrophils or HL-60 cells to: 1) purified P-selectin, 2) thrombin-stimulated platelets, and 3) phorbol 12-myristate 13-acetate-activated endothelial cells. These results suggest that the sialoglycoprotein recognized by this panel of monoclonal antibodies may function as a cell surface ligand for P-selectin.

Specific inhibition of leukotriene B4 (LTB4)-induced neutrophil emigration by 20-hydroxy LTB4: implications for the regulation of inflammatory responses.

1. The interaction between leukotriene B4 (LTB4) and its metabolite, 20-hydroxy LTB4 in the control of neutrophil emigration was examined in guinea-pig skin. 2. Leukotriene B4 (10-300 ng) elicited a dose-dependent increase in neutrophil infiltration (as measured by myeloperoxidase activity) 4 h after injection into guinea-pig skin. In contrast, 20-hydroxy LTB4 (30-1000 ng) displayed only weak inflammatory activity in this assay. 3. Although 20-hydroxy LTB4 had low agonist activity, this metabolite caused a potent dose-dependent inhibition of responses to LTB4 (100 ng), when administered systemically (ED50 = 1.3 micrograms kg-1, s.c.) without significantly affecting neutrophil infiltration in response to C5a (2 micrograms). Systemic administration of 20-carboxy LTB4 (10 micrograms) did not affect neutrophil accumulation in response to LTB4 or C5a. In addition, neither 15(S)-hydroxy 5(S)-HPETE(10 micrograms) nor lipoxin A4 (10 micrograms) inhibited responses to LTB4. 4. Addition of 20-hydroxy LTB4 (10(-11)-10(-8) M) to human blood prior to isolation of the neutrophils led to concentration-dependent decrease in the number of LTB4 receptors and decreased chemotactic responsiveness to LTB4 without affecting responses to C5a. Incubation of blood with 20-carboxy LTB4 (10(-8) M) did not reduce LTB4 receptor number of chemotactic responsiveness to LTB4. 5. These data indicate that although 20-hydroxy LTB4 is a weak agonist at LTB4 receptors, it can desensitize neutrophils to the effects of LTB4 via down-regulation of the high affinity receptor and thus provides evidence for a mechanism whereby inflammatory responses may be regulated.

A convenient cloning vector containing the GAL4 DNA-binding domain.

A DNA fragment encoding the yeast GAL4 DNA-binding domain (amino acids 3-147) was cloned into a convenient vector. This vector contains unique restriction sites at both the 5' and 3' ends and allows the generation of fusion proteins containing the GAL4 DNA-binding domain. These fusion proteins can be tested for their ability to activate transcription.

Inhibition of the mutant p53 gene in transformation assays.

Mutation of the p53 gene is a key element in the development of several human cancers. Intron 4, a noncoding region of the p53 gene, is required for optimal expression of that gene. We have previously shown that nuclear protein binds intron 4 and have defined the protein-binding site. In this paper we address the question, "Does the mutant p53 gene's ability to transform cells to the malignant phenotype depend on protein binding to intron 4?" Using an in vitro assay in which the mutant p53 gene and Ha-ras oncogene cooperate in transformation of cells to the malignant phenotype, we determined the ability of mutant mouse p53 gene constructs, with and without two base pair substitutions at the intron 4 protein-binding site, to participate in malignant transformation. On Day 1, 5 x 10(5) rat embryo fibroblasts were transfected by the calcium phosphate procedure with 10 micrograms of both a mutant p53 gene construct and Ha-ras oncogene. Malignant transformation was evidenced by the formation of discrete foci of heaped-up cells. After 14 days of incubation at 37 degrees C in DMEM and 10% fetal calf serum (8% CO2), the cells were stained with cresyl violet and the foci counted. In three separate experiments, the presence of two base pair substitutions at the intron 4 protein-binding site caused a significant decrease in the number of foci formed (P less than 0.05).

Analysis of p53 mutants for transcriptional activity.

The wild-type p53 protein functions to suppress transformation, but numerous mutant p53 proteins are transformation competent. To examine the role of p53 as a transcription factor, we made fusion proteins containing human or mouse p53 sequences fused to the DNA binding domain of a known transcription factor, GAL4. Human and mouse wild-type p53/GAL4 specifically transactivated expression of a chloramphenicol acetyltransferase reporter in HeLa, CHO, and NIH 3T3 cells. Several mutant p53 proteins, including a mouse p53 mutant which is temperature sensitive for suppression, were also analyzed. A p53/GAL4 fusion protein with this mutation was also transcriptionally active only at the permissive temperature. Another mutant p53/GAL4 fusion protein analyzed mimics the mutation inherited in Li-Fraumeni patients. This fusion protein was as active as wild-type p53/GAL4 in our assay. Two human p53 mutants that arose from alterations of the p53 gene in colorectal carcinomas were 30- to 40-fold less effective at activating transcription than wild-type p53/GAL4 fusion proteins. Thus, functional wild-type p53/GAL4 fusion proteins activate transcription, while several transformation competent mutants do so poorly or not at all. Only one mutant p53/GAL4 fusion protein remained transcriptionally active.

An intron binding protein is required for transformation ability of p53.

Regulatory elements in intron sequences have been identified for several eukaryotic genes. The fourth intron of p53 is known to increase expression of p53 in a position dependent manner. We asked whether p53 intron 4 sequences interacted with DNA binding proteins to exact their effect. Three overlapping DNA fragments spanning the 5' end of p53 intron 4 were determined to specifically interact with protein in nuclear extracts from several cell lines by band shift analysis. Methylation interference experiments were used to identify purine residues involved in this protein-DNA interaction. Two G nucleotides were identified at intron 4 positions 33 and 44 and these were replaced by T and C, respectively. These two single base pair substitutions in the intron resulted in 1) lack of protein binding and 2) decreased expression of p53 as measured by a transformation assay. Thus the binding of protein to p53 intron 4 was shown to have functional significance. These experiments demonstrated a specific protein binding region in the 5' end of intron 4 critical for p53 expression and distinct from those elements already known to be involved in splicing.

Transcriptional activation by wild-type but not transforming mutants of the p53 anti-oncogene.

The protein encoded by the wild-type p53 proto-oncogene has been shown to suppress transformation, whereas certain mutations that alter p53 become transformation competent. Fusion proteins between p53 and the GAL4 DNA binding domain were made to anchor p53 to a DNA target sequence and to allow measurement of transcriptional activation of a reporter plasmid. The wild-type p53 stimulated transcription in this assay, but two transforming mutations in p53 were unable to act as transcriptional activators. Therefore, p53 can activate transcription, and transformation-activating mutations result in a loss of function of the p53 protein. The inability of the p53 mutant proteins to activate transcription may enable them to be transformation competent.