Willem Van Dijk

Glycosylation of three molecular forms of human α1-acid glycoprotein having different interactions with concanavalin A

Human alpha 1-acid glycoprotein (AGP) was separated into a non-bound (AGP-A; 46%), a retarded (AGP-B; 39%) and a bound fraction (AGP-C; 15%) using concanavalin A (ConA)-Sepharose chromatography. The apparent molecular masses, as determined by SDS-PAGE, of the three fractions were 43.5, 42.3 and 41.2 kDa, respectively. The occurrence of N-linked di-, tri- and tetraantennary glycans on these three molecular forms (AGP-A, -B, and -C) was studied by sequential lectin-affinity chromatography of the 14C-labelled glycopeptides. These were obtained by extensive pronase treatment followed by N-[14C]acetylation of the peptide moieties. The glycopeptides of AGP-A did not bind to ConA-Sepharose whereas for AGP-B and AGP-C 18% and 44%, respectively, of the glycopeptides were bound as diantennary structures. Glycopeptide fractions of all three forms of AGP which were not bound to ConA-Sepharose were shown to contain equal amounts of both tri- and tetraantennary glycans by chromatography with Phaseolus vulgaris leukoagglutinating lectin (L-PHA). With the assumption that each molecule contains five glycosylation sites, it could be shown that AGP-A contains no diantennary structures whereas AGP-B and AGP-C contain one and two diantennary structures, respectively. In addition each of the molecular forms contains equal amounts of tri- and tetraantennary structures on the remaining glycosylation sites. The results of this study, therefore, exclude a uniformity of glycan chains in the three molecular forms of AGP. The degree of sialylation of each of the molecular forms was investigated by chromatography on L-PHA-agarose and Ricinus communis agglutinin-I--agarose both before and after desialylation of the glycopeptides. It was shown that about 90% of the biantennary glycans of both AGP-B and AGP-C were disialylated while the remainder were monosialylated. The degree of sialylation of the tri- and tetraantennary glycans was identical for the three molecular forms. In each case, one or more terminal galactose residues occurred on at least 20% of the tri- and 65% of the tetraantennary chains. It is suggested that the decrease in the exposure of galactose residues from AGP-A to AGP-C is related to the concomittant decrease in branching of the glycans of the three molecular forms. The relevance of these findings to studies on the function of AGP during inflammatory and liver diseases is discussed.

Lol p XI, a new major grass pollen allergen, is a member of a family of soybean trypsin inhibitor-related proteins

BACKGROUND Monoclonal antibodies were obtained against an unknown allergen from Lolium perenne grass pollen. The allergen had an apparent molecular mass of 18 kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Earlier immunoblotting studies had shown that carbohydrate-specific IgG antibodies recognize an antigen of similar size. OBJECTIVE We sought to characterize the allergen biochemically and immunologically. METHODS The amino acid sequence of the allergen was determined by automated Edman degradation, and its monosaccharide composition was determined by gas chromatographic analysis. A panel of 270 grass pollen-positive sera was assessed in a RAST with the purified allergen. Protease digestion (proteinase K) and chemical deglycosylation (trifluoromethane sulfonic acid) were used to distinguish between carbohydrate and peptide epitopes for IgE antibodies. RESULTS The allergen was shown to be a glycoprotein with a molecular mass of 16 kd, of which 8% is carbohydrate. Its amino acid sequence shares 32% homology with soybean trypsin inhibitor (Kunitz) but lacks its active site. No homology was found with known grass pollen allergens, hence it was designated Lol p XI. A high degree of homology (44%) was found with a tree pollen allergen, Ole e I, the major allergen of olive pollen. More than 65% of grass pollen-positive sera had IgE against Lol p XI. IgE reactivity was demonstrated both with the carbohydrate moiety and the peptide backbone. CONCLUSIONS Lol p XI is a new major grass pollen allergen carrying an IgE-binding carbohydrate determinant. Lol p XI is structurally related to the major allergen from olive pollen.

α1‐Acid glycoprotein fucosylation as a marker of carcinoma progression and prognosis

Serum α1‐acid glycoprotein (AGP), an acute‐phase protein secreted by the liver, carries α(1,3)‐fucosylated structures on its 5 highly branched, N‐linked sugar chains.

Con A-nonreactive human α1-acid glycoprotein (AGP) is more effective in modulation of lymphocyte proliferation than Con A-reactive AGP serum variants

Humanα1-acid glycoprotein (AGP) has been shown to modulate various cellular and humoral immune reactions in vitro. Using glycosidase-modified derivatives of AGP, the importance of its carbohydrate moiety with regard to these effects has been noted. In normal serum, three molecular AGP forms interacting differently with concanavalin A (Con A) are present. The ratio of these forms is often changed during various physiopathological conditions. In this study, we could show that differences exist between the three AGP forms with regard to their immunomodulatory effectiveness. At physiological concentrations, the Con A-nonreactive variant AGP-A induced a stronger inhibition of the anti-CD3 stimulated lymphocyte proliferation than the other forms. Interestingly, AGP-A was also found to be responsible for the stimulation of lymphocyte proliferation induced by low AGP concentrations in vitro. Both immunomodulatory effects of AGP were abrogated by desialylation of the glycoprotein. These results support an immunomodulatory role of AGP in conditions characterized by a changed ratio of the differently glycosylated AGP forms.

Glycosylation ofα1 glycoprotein in septic shock: Changes in degree of branching and in expression of sialyl Lewisx groups

The occurrence of differences in acute-phase response, with respect to concentration and glycosylation of α1-acid glycoprotein (AGP) was studied in the sera of patients surviving or not from septic shock. Crossed affino-immunoelectrophoresis was used with concanavalin A andAleuria aurantia lectin for the detection of the degree of branching and fucosylation, respectively, and the monoclonal CSLEX-1 for the detection of sialyl Lewisx (SLeX) groups on AGP. Septic shock apparently induced an acute-phase response as indicated by the increased serum levels and changed glycosylation of AGP. In the survivor group a transient increase in diantennary glycan content was accompanied by a gradually increasing fucosylation and SLeX expression, comparable to those observed in the early phase of an acute-inflammatory response. Remarkably, in the non-survivor group a modest increase in diantennary glycan content was accompanied by a strong elevation of the fucosylation of AGP and the expression of SLeX groups on AGP, typical for the late phase of an acute-phase response. Our results suggest that these changes in glycosylation of AGP can have a prognostic value for the outcome of septic shock.

Changes in glycosylation of acute-phase proteins in health and disease: Occurrence, regulation and function

The pathophysiological variations in different glycoforms of acute-phase glycoproteins in serum most likely result from changes in the glycosylation process during their biosynthesis in the parenchymal cells of the liver. Biosynthesis in other cells or tissues may contribute, but in general appears to play a minor role. Inflammatory cytokines appear to regulate the process, but glycosylation changes are independent of protein synthesis. In addition, other humoral factors such as corticosteroids and growth factors are involved. The interplay of these factors is determined by the stage of the disease (e.g rheumatoid arthritis), the physiological situation (e.g. pregnancy), or directly or indirectly by extraneous factors such as drugs (e.g. ethanol). Information about the functional implications of the changes is limited, but some reports suggest that for α1-acid glycoprotein the changes might affect the operation of the immune system.

Inflammation-induced expression of sialyl Lewisx is not restricted to α1-acid glycoprotein but also occurs to a lesser extent on α1-antichymotrypsin and haptoglobin

Acute and chronic inflammation-induced expression of sialyl Lewisx has already been shown to occur on α1-acid glycoprotein. We now demonstrate that this phenomenon is not restricted to α1-acid glycoprotein but also occurs on two other acute-phase proteins. ie on α-antichymotrypsin and on haptoglobin. The level of expression of sialyl Lewisx on these proteins was lower than on α1-acid glycoprotein, in all likelihood because α1-acid glycoprotein is the only acute-phase protein containing tetraantennary glycans. No expression of sialyl Lewisx was detectable on α1-protease inhibitor, a protein with a high diantennary glycan content. Non-sialylated Lewisx was not detectable on these major acute-phase proteins in any of the conditions studied. This indicates that the majority of the α3-linked fucose residues are present as sialyl Lewisx on α1-acid glycoprotein, α1-antichymotrypsin and haptoglobin. The absolute contribution to the total phenotype in plasma of protein containing this determinant in a multivalent form was highest for α1-acid glycoprotein. This leads us to propose that α1-acid glycoprotein is, among the acute-phase proteins studied, the one with the highest potential for interference with the extravasation of leukocytes by binding to the selectins.

Activation of human endothelial cells by tumor necrosis factor-alpha results in profound changes in the expression of glycosylation-related genes.

The endothelium plays a central role in the logistics of the immune system by allowing the selective transmigration of leukocytes, as well as the maintenance of the circulation and coagulation homeostasis. Evidence is increasing that the carbohydrate composition of the endothelial cell surface is critical for the cells to exert their physiological function. The major aim of this study is to unravel the mechanisms underlying the expression of carbohydrate structures by endothelial cells, which are involved in leukocyte adhesion and migration. Using quantitative real‐time PCR, the expression profile of a selected group of 74 glycosylation‐related genes has been determined in human umbilical vein endothelial cells (HUVEC) and human foreskin microvascular endothelial cells (FMVEC) under control and TNFα‐induced conditions. The set of genes comprised 59 glycosyltransferases, 6 mannosidases and 9 sulfotransferases. In parallel, the overall cell surface glycan profile has been assessed by the use of glycan‐specific lectins and monoclonal antibodies. The results demonstrate that HUVEC and FMVEC differ substantially in the expression of glycosylation‐related genes and, accordingly, also in the presence of different glycan epitopes on the cell membrane. Induction of an inflamed phenotype of the cells by treatment with TNFα differentially modulates a set of these genes in HUVEC and FMVEC resulting in a change in the cell membrane associated glycans that are of importance in inflammation‐related endothelial cell‐surface processes.

The Utility of Lipid-Associated Sialic Acid (LASA or LSA) as a Serum Marker for Malignancy

The utility of the lipid-associated sialic acid (LASA or LSA) test as a serum marker for malignancy is reviewed. The name LASA or LSA test is confusing because it suggests that only or mainly lipid-bound sialic acid is measured. In reality, glycoprotein-bound sialic acid is determined predominantly. The assay appears to have a particularly high positivity rate in leukemia, Hodgkins disease, melanoma, sarcoma, advanced ovarian carcinoma and oropharyngeal tumors, suggesting that LASA may serve as a valuable marker in these malignancies. As a consequence of the rise of sialic acid-rich acute-phase proteins, such as alpha 1-acid glycoprotein, in inflammatory diseases the specificity of LASA and therefore its diagnostic accuracy is low. LASA can be useful for monitoring cancer patients during treatment, especially in combination with other tumor markers.

Convergent Actions of IκB Kinase β and Protein Kinase Cδ Modulate mRNA Stability through Phosphorylation of 14-3-3β Complexed with Tristetraprolin

ABSTRACT Regulation of gene expression at the level of mRNA stability is a major topic of research; however, knowledge about the regulatory mechanisms affecting the binding and function of AU-rich element (ARE)-binding proteins (AUBPs) in response to extracellular signals is minimal. The β1,4-galactosyltransferase 1 (β4GalT1) gene enabled us to study the mechanisms involved in binding of tristetraprolin (TTP) as the stability of its mRNA is regulated solely through one ARE bound by TTP in resting human umbilical vein endothelial cells. Here, we provide evidence that the complex formation of TTP with 14-3-3β is required to bind β4GalT1 mRNA and promote its decay. Furthermore, upon tumor necrosis factor alpha stimulation, the activation of both Iκβ kinase and protein kinase Cδ is involved in the phosphorylation of 14-3-3β on two serine residues, paralleled by release of binding of TTP and 14-3-3β from β4GalT1 mRNA, nuclear sequestration of TTP, and β4GalT1 mRNA stabilization. Thus, a key mechanism regulating mRNA binding and function of the destabilizing AUBP TTP involves the phosphorylation status of 14-3-3β.