Multiple forms of selenoprotein P in a candidate human plasma standard reference material

Abstract

Selenoprotein P (SePP) is a unique selenium-containing protein responsible for the transport and distribution of the essential trace element selenium (Se) through the human body with the concentration of SePP in human blood representing the most useful marker of Se nutritional status. Although SePP has been extensively studied, the structure of SePP in human plasma remains unresolved. Two potential isoforms of SePP have been identified by Western blot analyses distinguished principally by differences in migration (51 kDa and 61 kDa). The biological relevance of the smaller isoform has been called into question by several studies reporting only one major SePP form (69 kDa) suggesting that the shorter 51 kDa is an artifact of protease activity during the SePP purification process. A deficiency of these Western blot analyses is that no information can be gleaned regarding the Se content of the potential isoforms. This study reports a characterization of SePP isoforms in a human plasma Standard Reference Material representative of a healthy US population. Following immunoprecipitation, three SePP isoforms were unequivocally identified at 45 kDa, 49 kDa and 57 kDa (termed as SePP45, SePP49 and SePP57) by LC-MS/MS analyses from a spectral searching approach. Selenium (Se) was detected by gel electrophoresis LA-ICP-MS in SePP49 and SePP57 which was confirmed by the identification of three selenopeptides covering the SePP sequence from residues 312-346 by LC-MS/MS analyses utilizing a sequence searching approach. Conversely, neither Se nor peptides covering SePP sequence from residues 306-346 was identified in SePP45 which suggests that SePP45 is a truncated isoform transcriptionally terminated at the 2nd in-frame UGA codon thereby terminating the protein with the Ser residue at position 299. An additional band at 23 kDa was found to contain Se but no peptides of SePP. Instead, glutathione peroxidase 3 (GPx3) was unequivocally identified within the band presumably being co-immunoprecipitated with the SePP providing preliminary evidence that SePP and GPx3 interaction may take place in vivo.

Selenium speciation in human serum and its implications for epidemiologic research: a cross-sectional study

“…There the reliability of our SePP determination was verified since the immunologically determined SePP concentration in a pooled CSF sample was 0.54 nM/l (±9.5%) with SePP being expressed as total protein. According to the literature data regarding SePP (10 Se atoms per SePP and molecular weight MW(SePP) = 61 kDa [32]) this SePP-protein concentration refers to 0.43 ± 0.04 g Se/l at SePP corresponding well to our value at 0.41 ± 0.01 g Se/l at SePP peak. Thus our method was verified by an independent reference method.…”

Sex-specific and inter-individual differences in biomarkers of selenium status identified by a calibrated ELISA for selenoprotein P

“…Accordingly, in the MS/MS-based analyses of the reference plasma SRM1950 of the U.S. population, three SELENOP molecules of 57, 49 and 45 kDa were identified [18]. Alternatively, slightly shortened SELENOP isoforms may result from a specific limited proteolysis within the Se-rich C-terminus [19].…”

“…However, there are indications that the circulating SELENOP is not a homogenous protein. Variants of different molecular weight have been described [13], [18], in relation to genotype, premature translational termination, limited posttranslational proteolysis or partial replacement of Sec by Cys [19], [20], [21], [22].…”

Selenium biochemistry and its role for human health

“…120 A recent study reported the separation and characterization of three distinct SelP isoforms also in human plasma, with Mw of 45, 49 and 57 kDa, the first being a truncated isoform lacking in Se. 121,122 SelP is mainly produced in the liver and then secreted into the plasma, where it incorporates the major part of Se, but it is expressed and probably secreted also by other tissues including the brain and the heart. As mentioned in Section 3.3, evidence supports functions of SelP in Se transport and homeostasis throughout the whole body.…”