Ake P. Elhammer

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

P-selectin glypoprotein 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), Galβ, 3GalNAc (36%), and Galβ, 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.

Properties of native and in vitro glycosylated forms of the glucagon-like peptide-1 receptor antagonist exendin (9–39)

The intestinal hormone glucagon-like peptide-1-(7-36)-amide (GLP-1) has recently been implicated as a possible therapeutic agent for the management of type 2 non-insulin-dependent diabetes mellitus (NIDDM). However, a major difficulty with the delivery of peptide-based agents is their short plasma half-life, mainly due to rapid serum clearance and proteolytic degradation. Using a peptide analog of GLP-1, the GLP-1 receptor antagonist exendin(9-39), we investigated whether the conjugation of a carbohydrate structure to exendin(9-39) would generate a peptide with intact biological activity and improved survival in circulation. The C-terminal portion of exendin(9-39) was reengineered to generate an efficient site for enzymatic O-glycosylation. The modified exendin(9-39) peptide (exe-M) was glycosylated by recombinant UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1) alone or in conjunction with a recombinant GalNAc alpha2,6-sialyltransferase (Sialyl-T), resulting in exe-M peptides containing either the monosaccharide GalNAc or the disaccharide NeuAc alpha2,6GalNAc. The nonglycosylated and glycosylated forms of exe-M competed with nearly equal potency (> 90% of control) with the binding of [125I]GLP-1 to human GLP-1 receptors expressed on stably transfected COS-7 cells. In addition, each peptide was equally effective for inhibiting GLP-1-induced cyclic adenosine monophosphate (cAMP) production in vitro. Studies with rats demonstrated that the modified and glycosylated forms of exendin(9-39) could antagonize exogenously administered GLP-1 in vivo. Interestingly, glycosylated exendin(9-39) homologs were more than twice as effective as the nonglycosylated peptide for inhibiting GLP-1-stimulated insulin production in vivo, suggesting a longer functional half-life in the circulation for glycosylated peptides. Results from in vivo studies with 3H-labeled peptides suggest that the glycosylated peptides may be less susceptible to modification in the circulation.

Cloning and molecular characterization of the gene encoding the Aureobasidin A biosynthesis complex in Aureobasidium pullulans BP-1938

The gene (aba1) encoding the NRPS complex responsible for the synthesis of the cyclic peptide antibiotic Aureobasidin A (AbA) in Aureobasidium pullulans BP-1938, was cloned using a combination of PCR and library screening approaches. The aba1 gene was found to consist of a single, intronless open reading frame (ORF) of 34,980 bp, encoding an 11,659 amino acid protein with a calculated molecular mass of 1,286,254 Da. Putative promoter and translation start elements were identified upstream from the putative ATG in the aba1 gene, and a consensus poly(A) addition signal (AATAAA) was identified 191 bp downstream of the translation termination codon (TGA). As predicted by the structure AbA, the aba1 gene encodes an enzyme composed of nine biosynthetic modules, eight of which contain adenylation domains with recognizable amino acid specificity-conferring code elements, and four of which contain embedded methylation domains. The biosynthetic module located at position one in the aba1 gene lacks recognizable specificity-conferring code elements, consistent with it being responsible for incorporation of the 2-hydroxy-3-methylpentanoic acid residue at that position in AbA. An unusual feature of the aba1 gene sequence is a very high degree of shared identity among eight of the biosynthetic modules, at both the nucleotide and amino acid level. The majority of the modules share better than 70% nucleotide identity with another module in the complex, and modules with the same amino acid adenylation specificity share up to 95% identity. Insertion of a hygromycin B phosphotransferase (HPT) gene cassette in place of the module 4 sequence in aba1 resulted in a cessation of AbA production, thus validating that the isolated gene encodes the AbA biosynthesis complex.

Generation of Broad-Spectrum Antifungal Drug Candidates from the Natural Product Compound Aureobasidin A.

The natural product aureobasidin A (AbA) is a potent, well-tolerated antifungal agent with robust efficacy in animals. Although native AbA is active against a number of fungi, it has little activity against Aspergillus fumigatus, an important human pathogen, and attempts to improve the activity against this organism by structural modifications have to date involved chemistries too complex for continued development. This report describes novel chemistry for the modification of AbA. The key step involves functionalization of the phenylalanine residues in the compound by iridium-catalyzed borylation. This is followed by displacement of the pinacol boron moiety to form the corresponding bromide or iodide and substitution by Suzuki biaryl coupling. The approach allows for synthesis of a truly wide range of derivatives and has produced compounds with A. fumigatus minimal inhibitory concentrations (MIC) of <0.5 μg/mL. The approach is readily adaptable to large-scale synthesis and industrial production.

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

ABSTRACTPSGL-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.