Anton Haselbeck

Structural characterization of glycoprotein carbohydrate chains by using digoxigenin-labeled lectins on blots

The carbohydrate structures of blotted glycoproteins can be analyzed by probing them with lectins. Here we describe a method where lectins conjugated with digoxigenin are used in combination with an anti-digoxigenin antibody AP conjugate as a very sensitive detection system for this type of analysis. The specificity of the lectins used, and the sensitivity of the detection system, provide valuable conclusions on the glycan structures. Only small amounts of glycoproteins are required for the analysis. The binding specificity of a set of lectins is demonstrated with various glycoproteins of defined carbohydrate structure. The application of these labeled lectins in combination with specific glycosidases for the characterization of the carbohydrate chains of recombinant tissue plasminogen activator and erythropoietin is presented.

O‐Glycosylation in Saccharomyces cerevisiae is initiated at the endoplasmic reticulum

The first mannose of O‐linked oligomannose chains in S. cerevisiae is transferred to Ser/Thr residues via dolichylphosphate mannose. Only this reaction (and not the subsequent reactions requiring GDP‐Man) proceeds at the endoplasmic reticulum.

Description and application of an immunological detection system for analyzing glycoproteins on blots

By introducing the steroid hapten digoxigenin specifically into sugars, a sensitive detection system for glycoproteins on blots has been developed. Sugars are oxidized to obtain aldehyde groups, which then react with digoxigenin-succinyl-ε-amido caproic acid hydrazide. A high-affinity antibody, conjugated to alkaline phosphatase, is used for the detection of the incorporated digoxigenin.This system allows the detection of nanogram-amounts of glycoproteins on blots, and its specificity allows a clear distinction of a glycoprotein from a non-glycoprotein. In combination with endo- and exoglycosidases it is very useful for determining the type of carbohydrate linkage in a glycoprotein, and by varying the oxidation conditions, specific labeling of sialic acids and terminal galactoses can be achieved.

Glycopeptide profiling of human urinary erythropoietin by matrix-assisted laser desorption/ionization mass spectrometry.

The site-specific glycan heterogeneity of human urinary erythropoietin was investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Owing to the small amount of protein available, a strategy combining optimal sensitivity and specificity was used. Erythropoietin was reduced, S-alkylated and digested with endoproteinase Lys C. The peptides were separated by reversed-phase high-performance liquid chromatography and the molecular masses of the peptides determined by MALDI-MS. The peptides were identified by comparing the experimental masses with the masses predicted from the cDNA derived amino acid sequence. Glycopeptides were identified from the mass spectra based on the peak pattern caused by the glycan heterogeneity. They were further characterized after treatment with neuraminidase and endoproteases. All N-glycosylation sites exhibited fucose-containing complex-type glycans. The N-glycosylation sites at Asn38 and Asn83 are mainly occupied by tetraantennary glycans, whereas Asn24 is occupied by a mixture of bi-, tri- and tetraantennary glycans. A molecular mass glycoprofile for each glycosylation site was established based on the relative peak intensities observed in the MALDI mass spectra of the desialylated glycopeptides.

Glycoprotein biosynthesis in Saccharomyces cerevisiae: ngd29, an N-glycosylation mutant allelic to och1 having a defect in the initiation of outer chain formation

Outer chain glycosylation in Saccharomyces cerevisiae leads to heterogeneous and immunogenic asparagine‐linked saccharide chains containing more than 50 mannose residues on secreted glycoproteins. Using a [3H]mannose suicide selection procedure a collection of N‐glycosylation defective mutants (designated ngd) was isolated. One mutant, ngd29, was found to have a defect in the initiation of the outer chain and displayed a temperature growth sensitivity at 37°C allowing the isolation of the corresponding gene by complementation. Cloning, sequencing and disruption of NGD29 showed that it is a non lethal gene and identical to OCH1. It complemented both the glycosylation and growth defect. Membranes isolated from an ngd29 disruptant or an ngd29mnn1 double mutant were no longer able, in contrast to membranes from wild type cells, to transfer mannose from GDPmannose to Man8GlcNAc2, the in vivo acceptor for building up the outer chain. Heterologous expression of glucose oxidase from Aspergillus niger in an ngd29mnn1 double mutant produced a secreted uniform glycoprotein with exclusively Man8GlcNAc2 structure that in wild type yeast is heavily hyperglycosylated. The data indicate that this mutant strain is a suitable host for the expression of recombinant glycoproteins from different origin in S. cerevisiae to obtain mammalian oligomannosidic type N‐linked carbohydrate chains.

Immunological detection of glycoproteins on blots based on labeling with digoxigenin

In the following, we describe the application of the DIG/antiDIG system for the (structural) analysis of glycoproteins on blots. Special emphasis is being placed on the variety of the different DIG applications in order to obtain structural information concerning the glycoprotein carbohydrate chains. Prominent among them is the use of lectins with well-known specificities for carbohydrate structures.

The Digoxigenin: Anti-Digoxigenin (DIG) System

The digoxigenin:anti-dioxigenin (DIG) indicator system is based on the specific interaction between the cardenolide steroid DIG, a chemically derived aglycon of digoxin and lanatoside C (see figure), and a high-affinity, DIG-specific antibody (Kessler, 1991).

Synthesis of retinylphosphate mannose in yeast and its possible involvement in lipid-linked oligosaccharide formation

A membrane fraction from Saccharomyces cerevisiae as well as a mannosyltransferase purified therefrom was shown to catalyze the transfer of mannose from GDPmannose to retinyl phosphate. The product formed has chromatographic and chemical properties characteristic for retinylphosphate mannose. The enzyme requires divalent cations. Mg2+ is more effective than Mn2+ with an optimum concentration around 25 mM. Amphomycin at a concentration of 0.1 mg/ml inhibits the reaction to 50%. Glycosyl transfer was specific for mannose residues from GDPmannose and did not occur with dolichylphosphate mannose nor with UDP galactose; UDPglucose is a poor donor. Formation of retinylphosphate mannose is inhibited by dolichyl phosphate. This observation as well as similarities between retinylphosphate mannose and dolichylphosphate mannose synthesis in respect to ion requirement, inhibition by amphomycin are suggestive that both reactions are catalyzed by one and the same enzyme. In experiments studying the glycosyl donor specificity in the assembly of lipid-linked oligosaccharide intermediates involved in N-glycosylation of proteins, it could be demonstrated that retinylphosphate mannose can replace dolichylphosphate mannose in the final steps of mannosylation.

Detection of Proteins and Glycoproteins on Western Blots

The analysis of proteins on blots after electrophoretic transfer from gels rather than analyzing the gels themselves is becoming increasingly popular due to several advantages, e.g., application of immunological techniques (“classical western blotting”); membranes do not shrink and are easy to handle; and several types of membranes with different advantages are available. However, general protein staining techniques of blots are by far not as well established as the very sensitive silver staining method of gels. Staining techniques using gold probes are approaching this sensitivity and are very useful [1]. By adapting the immunological digoxigenin (DIG)/anti-digoxigenin:alkaline phosphate ( :AP) system for the general staining of proteins on blots, we developed a second very sensitive system which should be even more useful due to its greater flexibility and convenient combination with other immunological detection methods of blots.

Blot Formats: Proteins and Glycoproteins

The analysis of proteins on blots after electrophoretic transfer from gels rather than analyzing the gels themselves is becoming increasingly popular due to several advantages, e.g., application of immunological techniques („classical western blotting“); membranes do not shrink and are easy to handle; and several types of membranes with different advantages are available. However, general protein staining techniques of blots are by far not as well established as the very sensitive silver staining method of gels. Staining techniques using gold probes are approaching this sensitivity and are very useful [1]. By adapting the immunological digoxigenin (DIG)/ anti-digoxigenin:alkaline phosphate ( :AP) system for the general staining of proteins on blots, we developed a second very sensitive system which should be even more useful due to its greater flexibility and convenient combination with other immunological detection methods of blots.