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Isolation and characterization of BetaM protein encoded by ATP1B4--a unique member of the Na,K-ATPase β-subunit gene family.

Abstract：

:ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a β-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase β-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was also characterized by SELDI-TOF mass spectrometry before and after deglycosylation. This allowed us to determine that the carbohydrate moiety of BetaM has molecular mass 5.9kDa and consists of short high-mannose type N-glycans. The results of direct analysis of the purified native eutherian BetaM protein provide first insights into structural properties underlying its entirely new evolutionarily acquired functions.

journal_name

Biochem Biophys Res Commun

journal_title

Biochemical and biophysical research communications

authors

Pestov NB,Zhao H,Basrur V,Modyanov NN

doi

10.1016/j.bbrc.2011.07.112

subject

Has Abstract

pub_date

2011-09-09 00:00:00

pages

543-8

issue

eissn

0006-291X

issn

1090-2104

pii

S0006-291X(11)01356-8

journal_volume

412

pub_type

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Isolation and characterization of BetaM protein encoded by ATP1B4--a unique member of the Na,K-ATPase β-subunit gene family.