Decreased sialylation of the acute phase protein alpha1-acid glycoprotein in feline infectious peritonitis (FIP)

Abstract

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).

Fig. 1

Purification of fAGP. (a) Q-Sepharose chromatography of feline serum. (b) S-Sepharose chromatography. Insert: SDS-PAGE of fAGP-containing protein fractions of each purification step. Molecular mass standards are shown in the first lane. Lane 2: fraction 2 from Q-Sepharose chromatography. Lane 4: fraction 4 from S-Sepharose chromatography. Lane WB: Western blotting of fractions 2 and 4 stained with anti-fAGP polyclonal antibody.

Fig. 2

Analysis of the glycosylation pattern of non-pathological fAGP. (a) Detection of oligosaccharide chains has been performed after Western blotting of 2 μg of pooled non-pathological fAGP. Staining was performed following alkaline phosphatase staining of a nitrocellulose membrane treated with—lane 1: SNAI, lane 2: MAA, lane 3: AAL and lane 4: ConA. (b) Density profile plot displaying the peaks corresponding to lane 1 (dotted line) and lane 2 (broken line). (c) Histograms displaying the average volume of density profile plots of non-pathological fAGP after reaction with SNAI and MAA. Value are expressed in relative pixel intensity.

Fig. 3

Analysis of the glycosylation pattern of pathological fAGP. Detection of oligosaccharide chains has been performed after Western blotting using the interaction with (a) SNAI, (b) MAA, (c) AAL and (d) ConA. np: 2 μg pooled non-pathologic fAGP, lanes 1–24: 2 μg each of fAGP from FIP affected cats. fAGP were stained as described in Section 2. The arrows on the right indicate the position of fAGP.

Fig. 4

Histograms displaying the average volume of density profile plots of non-pathological fAGP compared with FIP affected cats (a) and with FIP exposed cats (b), after reaction with SNAI and MAA. Value are expressed in relative pixel intensity.