W. van Dijk

Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors.

Pentameric ligand gated ion-channels, or Cys-loop receptors, mediate rapid chemical transmission of signals. This superfamily of allosteric transmembrane proteins includes the nicotinic acetylcholine (nAChR), serotonin 5-HT3, γ-aminobutyric-acid (GABAA and GABAC) and glycine receptors. Biochemical and electrophysiological information on the prototypic nAChRs is abundant but structural data at atomic resolution have been missing. Here we present the crystal structure of molluscan acetylcholine-binding protein (AChBP), a structural and functional homologue of the amino-terminal ligand-binding domain of an nAChR α-subunit. In the AChBP homopentamer, the protomers have an immunoglobulin-like topology. Ligand-binding sites are located at each of five subunit interfaces and contain residues contributed by biochemically determined ‘loops’ A to F. The subunit interfaces are highly variable within the ion-channel family, whereas the conserved residues stabilize the protomer fold. This AChBP structure is relevant for the development of drugs against, for example, Alzheimer’s disease and nicotine addiction.

Plasma concentration of C-reactive protein is increased in type I diabetic patients without clinical macroangiopathy and correlates with markers of endothelial dysfunction: evidence for chronic inflammation.

Summary Moderately increased plasma concentrations of C-reactive protein are associated with an increased risk of cardiovascular disease. C-reactive protein, its relation to a low degree of inflammatory activation and its association with activation of the endothelium have not been systematically investigated in Type I (insulin-dependent) diabetes mellitus. C-reactive protein concentrations were measured in 40 non-smoking patients with Type I diabetes without symptoms of macrovascular disease and in healthy control subjects, and in a second group of Type I diabetic patients (n = 60) with normo- (n = 20), micro- (n = 20) or macroalbuminuria (n = 20). Differences in glycosylation of α1-acid glycoprotein were assayed by crossed affinity immunoelectrophoresis. Activation of the endothelium was measured with plasma concentrations of endothelial cell markers. The median plasma concentration of C-reactive protein was higher in Type I diabetic patients compared with healthy control subjects [1.20 (0.06–21.64) vs 0.51 (0.04–9.44) mg/l; p < 0.02]. The Type I diabetic subjects had a significantly increased relative amount of fucosylated α1-acid glycoprotein (79 ± 12 % vs 69 ± 14 % in the healthy control subjects; p < 0.005), indicating a chronic hepatic inflammatory response. In the Type I diabetic group, log(C-reactive protein) correlated significantly with von Willebrand factor (r = 0.439, p < 0.005) and vascular cell adhesion molecule-1 (r = 0.384, p < 0.02), but not with sE-selectin (r = 0.008, p = 0.96). In the second group of Type I diabetic patients, increased urinary albumin excretion was associated with a significant increase of von Willebrand factor (p < 0.0005) and C-reactive protein (p = 0.003), which were strongly correlated (r = 0.53, p < 0.0005). Plasma concentrations of C-reactive protein were higher in Type I diabetic patients without (clinical) macroangiopathy than in control subjects, probably due to a chronic hepatic inflammatory response. The correlation of C-reactive protein with markers of endothelial dysfunction suggests a relation between activation of the endothelium and chronic inflammation. [Diabetologia (1999) 42: 351–357]

α1-Acid glycoprotein (orosomucoid): pathophysiological changes in glycosylation in relation to its function

The aim of this review is to summarize the research efforts of the last two decades with respect to (i) the determination and characterization of the changes in glycosylation of AGP under various physiological and pathological states; and (ii) the effects of such changes on its possible anti-inflammatory functions. It will become clear that the heterogeneity observed in the glycosylation of AGP in serum, represents various so-called glycoforms of AGP, of which the relative amounts are strictly determined by the (patho) physiological conditions.

α1-Acid Glycoprotein

α1 -Acid glycoprotein (AGP) is a heavily glycosylated serum glycoprotein containing five complex-type N-linked glycans (Fig. 1) [1,2]. It is a positive acute-phase glycoprotein of which the plasma level will increase 2-6-fold under acute (e.g. trauma, burns, bacterial infections) or chronic inflammatory conditions (e.g. rheumatoid arthritis (RA)) as a result of the hepatic acute-phase reaction. Its exact function is not known yet, although drug- and steroid-binding activities have been described [3,4], but also a number of immunomodulating activities [5–8]. The latter activities have appeared to depend on the carbohydrate moiety of the molecule. At least 12 glycoforms of AGP can be detected in normal serum [9]. They differ in degree of branching (di-antennary versus tri- or tetraantennary glycans) as well as in degree of fucosylation and sialylation of the glycans. Formerly, it was assumed that the occurrence of these glycoforms resulted from a non-specific heterogeneity in the glycosylation process of AGP during its biosynthesis in the liver. However, in the last decade this view has changed, because it has become clear that the relative occurrence of the AGP glycoforms in plasma is strongly dependent on the (patho) physiological condition. For example, acute inflammation induces transient time-dependent increases in the level of diantennary containing glycoforms, coinciding partially with abundant increases of strongly fucosylated glycoforms of AGP [10]. Inflammatory cytokines and hormones are involved in the regulation of these changes by an, as yet, unknown effect on the glycosylation process in the liver [11]. This knowledge has become available owing to the technique developed by Bog-Hansen [12] of crossed affmo-immunoelectrophoresis (CAIE) of sera or cell culture media with lectins as affino-component in the first dimension gel.

Do antimetabolites interfere with the glycosylation of cellular glycoconjugates

INTRODUCTION ANTIMETABOLITES are frequently used for the treatment of various neoplastic diseases, including solid tumors and leukemias [l], but the use of these compounds is limited by the occurrence of myeloid and gastrointestinal toxicity. For most of these drugs the mechanism of action has been investigated extensively in vitro and in viva [l-5]. These studies, in general, point to interference in purine or pyrimidine biosynthesis (cf. Table 1) leading to inhibition of DNA and/or RNA synthesis in dividing cells. Additional mechanisms, however, might be responsible for the cytotoxic effects on non-dividing cells, i.e. the majority of normal cells and tumor cells which are in resting phase. Several antimetabolites interfere with the manufacture of nucleotides (UTP, GTP and CTP) that are substrates for the manufacture of nucleotide sugars. Since nucleotide sugars are the precursors for the biosynthesis of glycoconjugates (cf. Fig. l), the glycosylation of both secreted and membrane-associated glycoconjugates might be affected in the presence of antimetabolites. The sugar chains extending from the cell surface are important determinants of the phenotype of cells [6] and have been implicated in a variety of cell surface interactions [6-lo]. Characteristic changes in cell surface oligosaccharides accompany normal and malignant development and hematopoietic differentiation [ll-221. Thus, for a variety of malignant cells a characteristic increase in molecular weight, caused by changes in branching [ 1 l-171 and/or increased amount of sialic acid [ 11, 12, 16-181, has been observed for cell surface oligosaccharide structures in comparison with those from non-malignant cells. The invasive capacity of cells [17, 19, 201 and the loss of binding of malignant cells to-the substratum [12, 211 is related positively to the observed increase in molecular weight. In the last decade the evidence is compelling that the variations in oligosaccharide structures observed coincide with changes in activities of specific glycosyltransferases [e.g. 14, 21, 23-261, indicating the importance of these enzymes in the regulation of the glycosylation process. The general occurrence of characteristic changes in glycoconjugates in relation to malignancy and differentiation gives sup-

Dental caries related to plasma IgG and alpha1-acid glycoprotein

This study was aimed at determining whether dental caries is associated with induction of the systemic immune system or cytokine response. For this purpose, 85 children from Den Pasar, Bali, Indonesia, aged 6–7 years, were examined clinically and blood plasma was obtained via finger puncture. The concentrations of the acute-phase protein α1-acid glycoprotein (AGP), total IgG and the specific IgG and IgM immunoglobulins against Streptococcus mutans were determined. Immunoelectrophoresis was used for the determination of the AGP concentration and ELISA for IgG and IgM detection. The mean dmft of the whole group was 8.8 ± 2.9, the mean number of infected pulps was 3.9 ± 2.2 and the mean number of abscesses was 0.5 ± 0.8. The plasma concentration of AGP ranged between 0.13 and 1.6 mg/ml serum (mean 0.86 ± 0.26 mg/ml). Stepwise regression analysis revealed that the concentration of IgG against S. mutans (log-transformed) was significantly correlated with dmft (adjusted r2 = 0.083, standardized β coefficient = 0.31, p = 0.008). When the concentration AGP was included in the model the correlation improved significantly (for IgG: adjusted r2 = 0.157, standardised β coefficient = 0.36, p = 0.002; for AGP: β coefficient = –0.30, p = 0.009). The results suggest a relationship between caries and systemic parameters of inflammation. On the basis of this, severe caries might have consequences on the general health of the subject.

Specific glycosylation of α1-acid glycoprotein characterises patients with familial Mediterranean fever and obligatory carriers of MEFV

BACKGROUND Familial Mediterranean fever (FMF) is a periodic febrile disorder, characterised by fever and serositis. The acute phase response during attacks of FMF results from the release of cytokines, which in turn induce increased expression and changed glycosylation of acute phase proteins. A recent study indicated that attacks in FMF are accompanied by a rise of plasma concentrations of serum amyloid A (SAA) and C reactive protein (CRP), which remain significantly raised during remission relative to healthy controls. Another study suggested that obligatory heterozygotes also display an inflammatory acute phase response. OBJECTIVE To determine the state of inflammation in homozygotic and heterozygotic MEFV genotypes. METHODS CRP and SAA were studied by enzyme linked immunosorbent assay (ELISA). The glycosylation of the acute phase protein, α1-acid glycoprotein (AGP), was visualised with crossed affinoimmunoelectrophoresis with concanavalin A as diantennary glycan-specific component and Aleuria aurantia lectin as fucose-specific affinity component. RESULTS FMF attacks were associated with an increase (p<0.05) in the serum inflammation parameters CRP, SAA, and AGP. The glycosylation of AGP showed an increase (p<0.05) in fucosylated AGP glycoforms, whereas the branching of the glycans remained unaffected. The glycosylation of AGP in the MEFV carrier group, compared with that in a healthy control group, was characterised by a significant increase (p<0.05) in branching of the glycans, whereas the fucosylation remained unaffected. CONCLUSION The findings suggest an FMF-specific release of cytokines, resulting in a different glycosylation of AGP between a homozygotic and heterozygotic MEFV genotype.

S15.16 Expression of (sialyl)-lewis x groups on human α1acid glycoprotein in acute and chronic inflammation

as mechanical obstruction and surfactant displacement by free fatty acids have been suggested. Recently the Human Lung Surfactant Protein SP-A has been characterized as one of several C-type lectins, which in the presence of Ca2*-ions bind specificially to certain carbohydrates. To test our hypothesis of specific lectin-carbohydrate interactions as part of the pathogenesis in meconium aspiration syndrome we set up to test the binding of native human SP-A towards meconium glycosphingolipids. Our data are in general agreement with recently published data (Childs, RA et al. (1992) J. Biol. Chem., 267, 9972-9979) and show a specific and Ca2+-related binding to two neutral glycosphingolipids present in meconium extract from all individuals tested; Galactosylceramide and Lactosylceramide. As was noted before, when using the thin layer chromatogram binding technique, the binding of SP-A to lactosylceramide species was strongest to those containing a-hydroxy fatty acids and phytosphingosine. In order to optimize a microtiter well binding assay and possibly reveal some factors of biological significance we have used a reduced factorial design of experimental approach. This includes in all 34 variables testing 69 different buffers with various concentrations of CaC12, NaC1, EDTA, pH, bovine serum albumin, NaN3 and Tween 20 at different steps of the assay as well as various concentrations, incubation times and incubation temperatures of blocking reagent, native SP-A, biotinylated anti SP-A antibody, ALP-coupled avidin and PNPP substrate. The use of this experimental design will be discussed.

Antimetabolite-induced increases in the invasive capacity of murine leukaemia L1210 cells

Pretreatment of murine leukaemia L1210 cells with non-lethal concentrations of various antimetabolites increased thein vitro invasive capacity of these cells into monolayers of rat embryo fibroblasts. The increase in invasive capacity was partly correlated with the induced cell cycle arrest. The concomitant increase in cell surface fucosylation and inhibition of invasion with sulphate indicate a role for glycoproteins in this process. Our results suggest that treatment with antimetabolites may lead to a more aggressive phenotype by altering cell surface properties.

How covalent and noncovalent interaction with SUMO can regulate the Ubl conjugation process

24 European Crystallographic Meeting, ECM24, Marrakech, 2007 Page s16 Acta Cryst. (2007). A63, s16 MS04 O1 How covalent and noncovalent interaction with SUMO can regulate the Ubl conjugation process Puck Knipscheer , Andrea Pichler, Willem J. van Dijk, Jesper V. Olsen, Matthias Mann, Frauke Melchior, Titia K. Sixma Netherlands Cancer Institute, Amsterdam, Max FPerutz Laboratories, Medical University Vienna, Max Planck Institute for Biochemistry, Martinried, Göttingen University , Italy. E-mail: t.sixma@nki.nl