Michael W. Spellman

Identification and structural analysis of the tetrasaccharide NeuAc.alpha.(2.fwdarw.6)Gal.beta.(1.fwdarw.4)GlcNAc.beta.(1.fwdarw.3)Fuc.alpha.1.fwdarw.O-linked to serine 61 of human factor IX

O-Linked fucose has been found attached to Thr/Ser residues within the sequence Cys-X-X-Gly-Gly-Thr/Ser-Cys in the N-terminal EGF domains of several coagulation/fibrinolytic proteins. Carbohydrate composition and mass spectrometric analyses of tryptic and thermolytic peptides containing the corresponding site (Ser-61) in the first EGF domain of human factor IX indicated the presence of a tetrasaccharide containing one residue each of sialic acid, galactose, N-acetylglucosamine, and fucose. The Ser-61 tetrasaccharide was not susceptible to alpha-fucosidase digestion. Fragments generated during mass spectrometric analysis indicated that fucose was the attachment sugar residue. The involvement of fucose in the carbohydrate-peptide linkage was confirmed by two-dimensional 1H NMR spectroscopic analysis of the glycopeptide containing factor IX residues 57-65. The complete structure of the tetrasaccharide was obtained by methylation analysis and two-dimensional 1H TOCSY and ROESY experiments as NeuAc alpha(2-->6)Gal beta(1-->4)GlcNAc beta(1-->3)Fuc alpha 1-->O-Ser61.

Study of the primary structure of recombinant tissue plasminogen activator by reversed-phase high-performance liquid chromatographic tryptic mapping

Two high-resolution tryptic maps have been developed for recombinant tissue plasminogen activator (rt-PA) that separate the expected 51 tryptic peptides. The trypsin digestion was performed after reduction and S-carboxymethylation of the protein. The high-performance liquid chromatographic separation of the tryptic peptides used a Nova-Pak C18 (5 microns) column with a mobile phase that contained 0.1% aqueous trifluoroacetic acid (TFA) or 50 mM sodium phosphate (pH 2.85) and a linear gradient of acetonitrile. A TFA solvent system was also used for re-purification and for characterization of the peptides isolated from the phosphate-based separation. All of the isolated peptides had compositions consistent with the sequence proposed for rt-PA. The identities of the glycopeptides were confirmed by lectin chromatography on concanavalin A-Sepharose. The mixture of tryptic peptides was also treated with endo-beta-N-acetylglucosaminidase H and peptide:N-glycosidase F to locate the position of either high mannose or complex oligosaccharides. These studies demonstrated that a high mannose oligosaccharide is attached to Asn-117 while complex carbohydrate side-chains are attached to Asn-184 and Asn-448. The residue Asn-184 is the site of optional glycosylation that results in the formation of two rt-PA variants that contain either two or three oligosaccharides.

Structure and Function in Recombinant HIV-1 gp120 and Speculation about the Disulfide Bonding in the gp120 Homologs of HIV-2 and SIV

The envelope glyco-proteins of the primate immunodeficiency viruses (HIV-1, HIV-2 and SIV) have been the objects of intense study since their discovery. The major envelope glycoprotein (gp120 in HIV-1) is of particular interest because it mediates the attachment of the virus to susceptible cells via the CD4 molecule1,2, it contains most of the important epitopes for neutralization of the virus by antibodies3,4,5, it plays an important role in the process by which the viral and host cell membranes fuse and the viral capsid gains access to the cytoplasm6,7, and its sequence variability is central to the ability of the virus to adapt to and escape the protective immune response of the host organism8. Complete understanding of these processes requires an understanding of the molecular structure of gp120 in detail. Such structural information has proven to be difficult to obtain because of the large size of gp120 (approximately 480 amino acids), its high degree of glycosylation (approximately 50% by weight), the high degree of heterogeneity of the oligosaccharides on the molecule, and the scarcity of material available for analysis.

IDENTIFICATION AND CHARACTERIZATION OF GLYCOPEPTIDES IN TRYPTIC MAPS BY HIGH-PH ANION-EXCHANGE CHROMATOGRAPHY

Publisher Summary This chapter describes the application of high-pH anion-exchange chromatography, with pulsed amperometric detection (HPAEC-PAD) to the identification and partial characterization of glycopeptides, within a reversed-phase high-performance liquid chromatography (HPLC) peptide map. HPAEC is a versatile chromatographic method that can be used for the detection and quantitation of monosaccharides, released by acid hydrolysis and for the analysis of intact oligosaccharides released enzymatically or chemically. A large number of other analytical methods, including mass spectrometry (MS), enzymatic microsequencing, nuclear magnetic resonance (NMR), and capillary electrophoresis, can also be applied to the characterization of glycopeptides; in general, a combination of several complementary analytical methods are needed to achieve a definitive carbohydrate structure determination. This chapter discusses the type of information that can be obtained, using a single, comparatively simple, analytical method.

Structural Characterization of a Novel Tetrasaccharide Attached to Ser-61 of Human Factor IX by Mass Spectrometry and 1H NMR Spectroscopy

Publisher Summary This chapter explores the structural characterization of a novel tetrasaccharide attached to ser-61 of human factor ix by mass spectrometry and 1H NMR spectroscopy. In a study described in the chapter, trypsin-digested S-carboxymethylated human plasma factor IX was prepared. No mass was observed that corresponded to any of the expected forms of the factor IX 44-80 tryptic peptide. The 44-80 peptide sequence is QYVDGDQCE S NPCLNGG S CK D DINSYECWCPFGFEGK. Tryptic RP-HPLC fractions were then subjected to amino acid analysis to identify a doublet peak containing the 44–80 peptides. The mass obtained for this fraction indicated the presence of some unknown modification that added 801.2 amu to the expected glycopeptide mass, far in excess of the 146.2 amu indicative of O-linked fucose. Digestion of the 44–80 peptides with thermolysin produced fragments demonstrating that the unknown -802 amu modification was found within residues 57–65. Digestion with α-fucosidase had no effect on the 57–65 glycopeptide, and the ES–MS fragmentation pattern indicated that fucose was present at the reducing end.