Proteomic analysis and immunodetection of the bovine milk osteopontin isoforms

Abstract

The aim of this study was to characterize the osteopontin (OPN) secreted in bovine milk and to determine whether the different forms are the product of spliced variants. Spliced variants of the human gene and secreted osteopontin isoforms have been reported in human tumor tissue. In bovine milk, we identified 2 major forms: one corresponding to the full-length coding transcript and a truncated version of this form. No alternative spliced transcripts were detected in the lactating mammary gland tissue, in milk somatic cells, or in peripheral blood immune cells. The 60-kDa bovine osteopontin (bOPN) and a truncated 40-kDa protein isoform were confirmed by mass spectrometry and further characterized by immunoblotting using a panel of 6 antibodies targeting different domains of the protein. Of the 3 human anti-OPN antibodies targeting the N-terminal segment of the protein, only one detected all forms on sodium dodecyl sulfate-PAGE; one human anti-OPN antibody failed to detect bOPN, whereas the other detected only the 60-kDa protein, albeit barely in its phosphorylated form. Detection was generally more sensitive when the 60-kDa protein was dephosphorylated. Two polyclonal antibodies raised against bOPN were tested: one targeting the milk-purified bOPN (bOPN-121) and one targeting a bovine epitope (synthetic peptide) corresponding to a carboxy-terminal domain of the protein (bOPN-117). The bOPN-121 antibody detected all forms irrespective of the phosphorylation status of bOPN. The bOPN-117 and the mouse anti-human OPN (hOPN-4) antibodies, which recognized different domains of the carboxy-terminal segment of the protein, also preferentially detected the dephosphorylated 60-kDa protein. Whereas phosphorylation had a major effect on detection for several antibodies, deglycosylation slightly decreased immunodetection for the tested antibodies. In particular, phosphorylation is the major posttranslational modification that influenced the weak detection capacity of several antibodies. This fact needs to be taken into account for immunodetection of milk content. In conclusion, the OPN forms secreted in bovine milk are not the product of alternative splicing. The 40-kDa protein appears to be a truncated hypophosphorylated variant of the full-length 60-kDa form, which is highly phosphorylated. Together, the proteomic and immunoblotting analyses used to characterize bovine milk OPN revealed the complex nature of the bovine milk OPN forms.

Genetic variations in the SPP1 promoter affect gene expression and the level of osteopontin secretion into bovine milk

“…Following SDS‐PAGE, immunodetection was performed as described previously (Bissonnette et al . ). The primary antibodies, MAB193P (Maine Biotechnology Services, Inc.) and custom‐made bOPN‐117 (Bissonnette et al .…”

“…The monoclonal antibody MAB193P recognizes one epitope located within the N‐terminal part of the OPN protein, and epitopes corresponding to the polyclonal antibody bOPN‐117 are located in the carboxyl side of the protein (Bissonnette et al . ). The milk and plasma samples were analyzed by sandwich ELISA.…”

“…The primary antibodies, MAB193P (Maine Biotechnology Services, Inc.) and custom‐made bOPN‐117 (Bissonnette et al . ), were diluted 1:1000 and 1:500 respectively. The monoclonal antibody MAB193P recognizes one epitope located within the N‐terminal part of the OPN protein, and epitopes corresponding to the polyclonal antibody bOPN‐117 are located in the carboxyl side of the protein (Bissonnette et al .…”

“…Subsequently, the polyclonal antibody bOPN‐121 (Bissonnette et al . ) diluted 1:500 in blocking buffer was added, and the plates were incubated at room temperature for 2 h. Following the washing step, a solution containing a horseradish peroxidase‐coupled antibody (anti‐rabbit IgG, NA934V; GE Healthcare Bio‐Sciences Inc.) diluted 1:10 000 in blocking buffer was added, and incubation was carried out at room temperature for 90 min. Following the final wash, the substrate, TMB Microwell Peroxidase Substrate (KPL, Inc.), was incubated for 10 min, and an equivalent volume of stopping solution (2.0 m H 2 SO 4 ) was added.…”

The Use of “Omics” in Lactation Research in Dairy Cows

“…In recent decades, proteomics has served as an attractive research approach to study milk protein expression in dairy cows, i.e., the milk fat globule membrane (MFGM) components and milk protein profile [76,79,96]. Reinhardt and Lippolis [76] studied MFGM in mid-lactation and identified 120 proteins that are mainly associated with cell signaling and membrane/protein trafficking.…”