Abstract
The serum protein α1-acid glycoprotein (AGP), also known as orosomucoid, is generally described as an archetypical positive acute phase protein. Here, porcine AGP was identified, purified and characterized from pooled pig serum. It was found to circulate as a single chain glycoprotein having an apparent molecular weight of 43 kDa by SDS-PAGE under reducing conditions, of which approximately 17 kDa were accounted for by N-bound oligosaccharides. Those data correspond well with the properties of the protein predicted from the single porcine AGP gene (ORM1, Q29014 (UniProt)), containing 5 putative glycosylation sites. A monoclonal antibody (MAb) was produced and shown to quantitatively and specifically react with all microheterogenous forms of pig AGP as analyzed by 2-D electrophoresis. This MAb was used to develop an immunoassay (ELISA) for quantification of AGP in pig serum samples. The adult serum concentrations of pig AGP were in the range of 1–3 mg/ml in a number of conventional pig breeds while it was lower in Göttingen and Ossabaw minipigs (in the 0.3 to 0.6 mg/ml range) and higher in young (2–5 days old) conventional pigs (mean: 6.6 mg/ml). Surprisingly, pig AGP was found to behave as a negative acute phase protein during a range of experimental infections and aseptic inflammation with significant decreases in serum concentration and in hepatic ORM1 expression during the acute phase response. To our knowledge this is the first description in any species of AGP being a negative acute phase protein.
Highlights
Alpha-1-acid glycoprotein (AGP), known as orosomucoid, is a remarkable serum protein, among the most glycosylated proteins in serum with 40–50% of its mass constituted by carbohydrate and having a very low isoelectric point due to its high content of sialic acid [1]
Unspecific reactions with a number of unrelated serum proteins were seen in salted-out serum, the identity of this band was confirmed by the identical position and appearance of purified pig AGP in reducing SDSPAGE and in Western blotting (Fig. 1A, lane 2, both panels)
Molecular heterogeneity was indicated in the first ion exchange chromatography step, as pig AGP was found both in the run-through from the CM Sepharose column and in eluted fractions obtained by increasing the pH of the buffer, corresponding to more acidic and less acidic isoforms, Figure 5
Summary
Alpha-1-acid glycoprotein (AGP), known as orosomucoid, is a remarkable serum protein, among the most glycosylated proteins in serum with 40–50% of its mass constituted by carbohydrate and having a very low isoelectric point due to its high content of sialic acid [1] It has a number of microheterogenous isoforms related to variations in its carbohydrate structure and sialic acid content which are both altered in various disease states (reviewed by [2]). Stone and Maurer (5) found that expression of pig AGP is developmentally regulated with high liver expression in the late stage foetus, decreasing 3–4 times in newborns and further dropping to approximately 100 times less than foetal abundance in the adult liver This confirms other reports describing the protein as constituting up to 50% of total serum protein in newborn pigs, decreasing approximately 30 times in the adult circulation [8], [9]. In addition to the identification of pig AGP in 2-D electrophoresis as a microheterogeneous acidic protein [7], a ConA-binding form of pig AGP in bronchoalveolar lavage fluid (BALF) being microheterogeneous with molecular weights in the range of 40–55 kDa and a range of isoelectric points around 3–4 has been described by [12]
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