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alpha(1)-Acid glycoprotein (AGP, orosomucoid) is a normal constituent of bovine blood. AGP is an immunocalin, a binding protein that can also exert several immunomodulatory functions. In this paper we investigated the effect of bovine alpha(1)-acid glycoprotein (boAGP) on spontaneous and staurosporine-induced apoptosis of blood derived monocytes purified using magnetic cell sorting techniques. Bovine AGP was purified from blood following a chromatographic protocol. The homogeneous protein was used to stimulate the cells as well to raise a polyclonal antibody, that was used throughout all the experiments. When monocytes were incubated with high concentrations of boAGP (0.9 mg/ml), similar to those found in bovine plasma during systemic reaction to inflammation, their spontaneous apoptosis rate was suppressed, as determined by caspase-3/7 enzymatic activity assay. Similar results were obtained when apoptosis was induced by adding staurosporine, a potent protein kinase inhibitor. The apoptosis-modulating activity of boAGP was dependent on its concentration, since physiological concentrations of boAGP (0.3 mg/ml) did not exhibit a statistically significative anti-apoptotic activity. We also investigated whether this apoptosis-modulating activity was dependent on the terminal sialic acid residues exposed on the surface of the protein. Enzymatic treatment with neuraminidase, that cleaves terminal sialic acid residues, completely abolished boAGP's anti-apoptotic activity. These results suggest that the protective effect of AGP is likely mediated by its sialic acid terminal groups.

Glycosylation is one of the most important post-translational modifications of proteins, and has been widely acknowledged as one of the most important ways to modulate both protein function and lifespan. The acute phase proteins are a major group of serum proteins whose concentration is altered during various pathophysiological conditions. The aim of this paper is to review the structure and functions of the alpha1-acid glycoprotein (AGP). AGP belongs to the subfamily of immunocalins, a group of binding proteins that also have immunomodulatory functions. One of the most interesting features of AGP is that its glycosylation microheterogeneity can be modified during diseases. This aspect is particularly remarkable, since both the immunomodulatory and the binding properties of AGP strongly depend on its carbohydrate composition. For these reasons, AGP can be considered an outstanding model for the study of glycan pattern modification during diseases. This review is focused on the most recent studies on the occurrence of different glycoforms in plasma and tissues and how the appearance of different oligosaccharide patterns during systemic inflammation or diseases can influence AGP's biological functions. The first part of the review will describe the structure of AGP and the several biological functions identified so far for this protein. The second part will be devoted to the post-translational modifications of the oligosaccharides micro-heterogeneity of AGP caused by pathological states. A critical evaluation of the impact of different AGP glycoforms on both its transport and anti-inflammatory features, and how the modifications of the glycan pattern can be utilized in clinical biochemistry, is also discussed.

International audience; $\alpha$1-acid glycoprotein (AGP) is an immunomodulatory protein expressed by hepatocytes in response to the systemic reaction that follows tissue damage caused by inflammation, infection or trauma. This paper presents the detection of bovine AGP (boAGP) in mammary secretions (colostrum and milk) and mammary gland tissue. Bovine AGP was detected by Western blotting in all the samples analysed, and could be quantified in colostrum at 162 (± 63.7) $\mu$g/mL and 114.5 (± 67.8) $\mu$g/mL during the first 12 h and 24 h respectively. In mature milk, the boAGP concentration clearly decreased and was no longer detectable using the Radial Immunodiffusion (RID) technique. The concentration of mature milk boAGP was therefore semi-quantified using an anion-exchange chromatographic procedure that allowed the concentration of the protein to be determined. The presence of AGP in bovine milk was confirmed by the internal sequence analysis performed following purification to homogeneity of the protein from milk. The concentration of AGP in bovine milk with low SCC (< 250 000) was very similar to that from bovine milk with high SCC (> 250 000). In order to investigate the origin of AGP in bovine milk, a search for mRNA was carried out in somatic cells and mammary gland tissue: mRNA expression of the boAGP gene was detected in mammary gland tissue, but not in somatic cells. Finally, the cDNA sequence of the boAGP was determined, and is hereby presented.

α1-Acid glycoprotein (AGP) is considered one of the major acute phase proteins in cats. In humans, AGP is a heavily glycosylated protein that undergoes several modifications of its glycan moiety during acute and chronic inflammatory pathologies. In this paper we present the feline AGPs (fAGP) glycan moiety modifications in the course of two prevalent feline diseases, the FIV (feline immunodeficiency virus) dependent feline acquired viral immunodeficiency and the feline leukemia virus (FeLV) associated lymphoma. The glycan moiety of fAGP was investigated by means of the binding of its oligosaccharides residues with specific lectins. Four lectins were used: Sambucus nigra agglutinin I and Maackia amurensis agglutinin lectins were used to detect sialic acid residues, Aleuria aurantia lectin was used to detect l -fucose residues and Concanavalin A was used to evaluate the degree of branching. It was found that fAGP undergoes several post-translational modifications of its glycan pattern: in particular the degree of sialylation is increased in FeLV-positive cats diagnosed with lymphoma, while FeLV-positive that did not presented any specific clinical signs cats do not present any increase of expression of sialic acid on the surface. Furthermore, FIV induced a modification of the glycan moiety of fAGP, which however varied widely among individuals. In order to determine the number and the position of oligosaccharide chains, the cDNA sequence of fAGP was also determined. The translation of the mature fAGP coding sequence gave rise to a sequence of 183 residues, with five potential N-glycosylation sites, but also with seven potential phosphorylation sites.

Feline infectious peritonitis (FIP) is an immune-mediated disease of domestic and exotic felides infected with feline coronavirus. FIP is characterized by the overexpression of an acute phase protein, the alpha1-acid glycoprotein (AGP). In humans, AGP is a heavily glycosylated protein that undergoes several modifications of its glycan moiety during acute and chronic inflammatory pathologies. We studied the changes in AGP glycosylation in the course of FIP. Specifically, we focussed our attention on the degree of sialylation, fucosylation and branching. This study presents a purification method for feline AGP (fAGP) from serum, using an ion exchange chromatography strategy. The glycosylation pattern was analyzed in detail by means of interaction of purified fAGP with specific lectins. In particular, Sambucus nigra agglutinin I and Maackia amurensis agglutinin lectins were used to detect sialic acid residues, Aleuria aurantia lectin was used to detect L-fucose residues and Concanavalin A was used to evaluate the branching degree. By this method we showed that fAGP did not present any L-fucose residues on its surface, and that its branching degree was very low, both in normal and in pathological conditions. In contrast, during FIP disease, fAGP underwent several modifications in the sialic acid content, including decreased expression of both alpha(2-6)-linked and alpha(2-3)-linked sialic acid (76 and 44%, respectively when compared to non-pathological feline AGP).

α1-Acid glycoprotein (AGP) is a lipocalin that is produced mainly by the liver and secreted into plasma in response to infections and injuries. In this study, we evaluated AGP isoforms that can be detected in bovine milk. We found that milk-AGP content is made up of at least two isoform groups, a low MW group (44 kDa) that is produced in the mammary gland (MG-AGP), and a higher MW group (55–70 kDa), that is produced by somatic cells (SC-AGP). Identical SC-AGP isoforms can be found both in milk and blood PMN cells. Analysis of the mammary tissue cDNA showed that the sequence of the MG-AGP isoform is identical to that of plasma AGP. Each group contains several proteins with different MWs and different isoelectric points, as shown by 2D-electrophoresis. The glycosylation patterns of these isoforms were analysed by means of specific lectin binding, to evaluate the degree of sialylation, fucosylation and branching. The MG-AGP glycan pattern was identical to plasma AGP produced by the liver. Several differences were detected, however, between plasma and SC-AGP isoforms, the most evident being the strong degree of fucosylation and the elevated number of di-antennary glycans in SC-AGP. Immunohistochemistry showed that AGP is found in all tissues that make up the mammary gland, but that it is most likely produced for the main part by the alveoli.