Hubert Krotkiewski

Galactosylation of IgG from rheumatoid arthritis (RA) patients – changes during therapy

It is well documented that serum IgG from rheumatoid arthritis (RA) patients exhibits decreased galactosylation of its conservative N-glycans (Asn-297) in CH2 domains of the heavy chains; it has been shown that this agalactosylation is proportional to disease severity. In the present investigation we analyzed galactosylation of IgG derived from the patients using a modified ELISA-plate test, biosensor BIAcore and total sugar analysis (GC-MS). For ELISA and BIAcore the binding of IgG preparations, purified from the patients’ sera, to two lectins: Ricinus communis (RCA-I) and Griffoniasimplicifolia (GSL-II) was applied. Based on ELISA-plate test an agalactosylation factor (AF, a relative ratio of GSL-II/RCA-I binding) was calculated, which was proportional to actual disease severity. Repeated testing of several patients before and after treatment with methotrexate (MTX) alone or in combination with Remicade (a chimeric antibody anti-TNF-α) supplied results indicating an increase of IgG galactosylation during the treatment. This introductory observation suggests that IgG galactosylation may be an additional indicator of the RA patients’ improvement.

The structure of glycophorins of animal erythrocytes

Glycophorins are red cell membrane sialoglycoproteins, which contain multipleO-linked oligosacchride chains and carry most of the cell surface sialic acid. Due to this high content of sialic acid the glycophorins are strongly stained with periodic acid-Schiff (PAS) reagent after sodium sodicylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The term “glycophorin” was proposed initially for human red cell sialoglycoproteins [1,2] and now it is also used for sialoglycoproteins in animal red cell membranes. Furthermore, similar glycoproteins of non-erythrocyte origin have also been identified and given the same name [3], although the terms “leukosialin” and “sialophorin” were proposed for a major sialoglycoproteins of human leukocytes [4,5]. In this article the term “glycophorin” will be used only for sialoglycoproteins existing in the erythrocyte membrane.Glycophorins of human erythrocytes, carrying blood group MN, Ss and other determinants, have been thoroughly studied and their properties described in several review articles [3,6,7,8]. The aim of this article is to summarize studies carriedout on the structure of non-human glycophorins, although some data concerning human glycophorins are included for comparative purposes.

Investigating the glycosylation of normal and ovarian cancer haptoglobins using digoxigenin-labelled lectins

Human haptoglobin (Hp), prepared from 10 normal sera and 10 ovarian cancer sera as well as from 11 ovarian cancer ascitic fluids, was characterized with regard to its reactivities with different lectins. Digoxigenin-labelled lectins [peanut agglutinin (PNA),Arachis hypogaea; SNA,Sambucus nigra; MAA,Maackia amurensis; DSA,Datura stramonium; and Con A, concanavalin A] with different carbohydrate specific moieties were used to identify sugar structures in Hp by blotting and by a quantitative assay in multiwell plates [lectin/enzyme-linked immunosorbent assay (ELISA)]. It was found that the lectin blotting was only useful for preliminary investigations, but that the lectin/ELISA gave interesting results that indicated the presence ofN-linked complex chains. Despite the fact that PNA interacted weakly with desialylated Hp in lectin blotting, no other evidence was obtained to suggest the presence ofO-linked glycans. Quantitative differences between normal and cancer Hp were observed for Con A, SNA and MAA, but no difference was found in the reaction with DSA. The binding of cancer Hp to Con A and SNA was twice that of normal Hp. Normal serum and ascitic fluid Hp bound similar amounts of MAA, but three times that observed for cancer serum Hp. Our results suggest that normal and ovarian cancer Hp differ in the content of carbohydrate structures containing sialic acid linked α(2–6) or α(2–3) to galactose and the type of oligosaccharide branching.

Muscle FBPase in a complex with muscle aldolase is insensitive to AMP inhibition

Real‐time interaction analysis, using the BIAcore biosensor, of rabbit muscle FBPase–aldolase complex revealed apparent binding constant [K Aapp] values of about 4.4×108 M−1. The stability of the complex was down‐regulated by the glycolytic intermediates dihydroxyacetone phosphate and fructose 6‐phosphate, and by the regulator of glycolysis and glyconeogenesis – fructose 2,6‐bisphosphate. FBPase in a complex with aldolase was entirely insensitive to inhibition by physiological concentrations of AMP (I 0.5 was 1.35 mM) and the cooperativity of the inhibition was not observed. The existence of an FBPase–aldolase complex that is insensitive to AMP inhibition explains the possibility of glycogen synthesis from carbohydrate precursors in vertebrates’ myocytes.

Overexpression of the Gene Encoding GTP:Mannose-1-Phosphate Guanyltransferase, mpg1, Increases Cellular GDP-Mannose Levels and Protein Mannosylation in Trichoderma reesei

ABSTRACT To elucidate the regulation and limiting factors in the glycosylation of secreted proteins, the mpg1 and dpm1 genes from Trichoderma reesei (Hypocrea jecorina) encoding GTP:α-d-mannose-1-phosphate guanyltransferase and dolichyl phosphate mannose synthase (DPMS), respectively, were overexpressed in T. reesei. No significant increases were observed in DPMS activity or protein secretion in dpm1-overexpressing transformants, whereas overexpression of mpg1 led to a twofold increase in GDP-mannose (GDPMan) levels. GDPMan was effectively utilized by mannnosyltransferases and resulted in hypermannosylation of secreted proteins in both N and O glycosylation. Overexpression of the mpg1 gene also increased the transcription of the dpm1 gene and DPMS activity. Our data indicate that the level of cellular GDPMan can play a major regulatory role in protein glycosylation in T. reesei.

Signaling and Regulatory Functions of Bioactive Sphingolipids as Therapeutic Targets in Multiple Sclerosis

Spingolipids (SLs) are an important component of central nervous system (CNS) myelin sheaths and affect the viability of brain cells (oligodendrocytes, neurons and astrocytes) that is determined by signaling mediated by bioactive sphingoids (lyso-SLs). Recent studies indicate that two lipids, ceramide and sphingosine 1-phosphate (S1P), are particularly involved in many human diseases including the autoimmune inflammatory demyelination of multiple sclerosis (MS). In this review we: (1) Discuss possible sources of ceramide in CNS; (2) Summarize the features of the metabolism of S1P and its downstream signaling through G-protein-coupled receptors; (3) Link perturbations in bioactive SLs metabolism to MS neurodegeneration and (4) Compile ceramide and S1P relationships to this process. In addition, we described recent preclinical and clinical trials of therapies targeting S1P signaling, including 2-amino-2-propane-1,3-diol hydrochloride (FTY720, fingolimod) as well as proposed intervention to specify critical SL levels that tilt balances of apoptotic/active ceramide versus anti-apoptotic/inactive dihydroceramide that may offer a novel and important therapeutic approach to MS.

Lumican: A new inhibitor of matrix metalloproteinase‐14 activity

We previously showed that lumican regulates MMP‐14 expression. The aim of this study was to compare the effect of lumican and decorin on MMP‐14 activity. In contrast to decorin, the glycosylated form of lumican was able to significantly decrease MMP‐14 activity in B16F1 melanoma cells. Our results suggest that a direct interaction occurs between lumican and MMP‐14. Lumican behaves as a competitive inhibitor which leads to a complete blocking of the activity of MMP‐14. It binds to the catalytic domain of MMP‐14 with moderate affinity (K D ∼ 275 nM). Lumican may protect collagen against MMP‐14 proteolysis, thus influencing cell–matrix interaction in tumor progression.

Structural analysis by fast-atom-bombardment mass spectrometry of the mixture of alditols derived from the O-linked oligosaccharides of murine glycophorins

The O-glycosylically linked oligosaccharides from glycophorins of BALB/c mouse erythrocytes were released as a mixture of alditol derivatives on reductive beta-elimination. A new approach, based on periodate oxidation in combination with f.a.b.-m.s., was used to elucidate the structure of one of the branched derivatives in the mixture. Evidence for the anomeric configuration was obtained by 500-MHz 1H-n.m.r. spectroscopy. The following structures were found: (Formula: see text).

An improved approach to the analysis of the structure of small oligosaccharides of glycoproteins: application to the O-linked oligosaccharides from human glycophorin A

Treatment of purified human glycophorin A with alkaline borohydride cleaved the oligosaccharide side chains to yield alditol derivatives that were separated by gel filtration into three mixtures of low molecular weight compounds. Each mixture was oxidised with periodate, and the products were reduced with borohydride and analysed after acetylation or methylation by GLC-MS and FABMS. The resulting data allowed the monosaccharide sequence and linkage positions to be assigned to each component of the mixtures. The anomeric configuration was determined by 1H NMR spectroscopy of the intact fractions. The structures of a desialylated tetrasaccharide, two monosialylated trisaccharides, and five other minor products were defined.

Overexpression of GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae corrects defects in dolichol-linked saccharide formation and protein glycosylation.

Thermosensitive mutants of Saccharomyces cerevisiae, affected in the endoplasmic reticulum (ER) located glycosylation, i.e. in Dol-P-Man synthase (dpm1), in beta-1,4 mannosyl transferase (alg1) and in alpha-1,3 mannosyltransferase (alg2), were used to assess the role of GDP-Man availability for the synthesis of dolichol-linked saccharides. The mutants were transformed with the yeast gene MPG1 (PSA1/VIG9) encoding GDP-Man pyrophosphorylase catalyzing the final step of GDP-Man formation. We found that overexpression of MPG1 allows growth at non-permissive temperature and leads to an increase in the cellular content of GDP-Man. In the alg1 and alg2 mutants, complemented with MPG1 gene, N-glycosylation of invertase was in part restored, to a degree comparable to that of the wild-type control. In the dpm1 mutant, the glycosylation reactions that depend on the formation of Dol-P-Man, i.e. elongation of Man(5)GlcNAc(2)-PP-Dol, O-mannosylation of chitinase and synthesis of GPI anchor were normal when MPG1 was overexpressed. Our data indicate that an increased level of GDP-Man is able to correct defects in mannosylation reactions ascribed to the ER and to the Golgi.