Molecular cloning and characterization of human age-related NADH oxidase (arNOX) proteins as members of the TM9 superfamily of transmembrane proteins

Age-related NADH oxidase (arNOX = ENOX3) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is present in blood and other body fluids. The activity was purified to apparent homogeneity from human urine and resolved by 2-D gel electrophoresis into a series of 24 to 32 kDa components of low isoelectric point. The purified proteins were resistant both to N-terminal sequencing and trypsin cleavage. Cleavage with pepsin revealed peptides corresponding to the TM9 family of transmembrane proteins. Peptide antisera raised to all five members of the human TM9 family sequentially blocked the arNOX activity of human saliva and sera. The soluble truncated N-terminus of the human homolog TM9SF4 was expressed in bacteria. The recombinant protein was characterized biochemically and exhibited ar-NOX activity. The findings identify five arNOX isoforms each of which correspond to one of the five known TM9 family members. The exfoliated soluble arNOX forms are derived from the 24 to 32 kDa N-termini exposed to the cell’s exterior at the cell surface. Each of the shed forms contain putative functional motifs characteristic of ECTO-NOX (ENOX) proteins despite only minimal sequence identity. Our findings identify arNOX as having functional characteristics of ENOX proteins and the TM9 superfamily of proteins as the genetic origins of the five known arNOX isoforms present in human sera, plasma and other body fluids1.

Age-Related Surface Oxidases Shed into Body Fluids as Targets to Prevent Skin Aging and Reduce Cardiovascular Risk

Age-related Ecto-Nicotinamide Adenine Dinucleotide Oxidase Disulfide Thiol Exchangers 3 (ENOX3) or age-related NADH oxidases (arNOX) are expressed at the cell surface as five members of the TM-9 superfamily, initially membrane anchored, all functionally similar, with the N-termini exposed at the cell’s exterior. ECTO-NOXes are cell surface proteins with both time-keeping CoQH2 [NAD(P)H] oxidase and protein disulfidethiol interchange activities. They are designated as ECTO-NOX proteins because of their localization on the outer surface of the plasma membrane and to distinguish them from the phox-NOXes of host defense. A ca. 30 kDa N-terminal fragment is cleaved and accumulates in body fluids (serum, saliva, urine, perspiration). arNOXes appear around age 30 and increase steadily thereafter. Reduced quinones, i.e., reduced coenzyme Q, of the plasma membrane are natural substrates. NAD(P)H is oxidized as an artificial substrate. In one phase of the arNOX cycle electrons are transferred to oxygen to generate superoxide. Substrates for the shed forms of arNOX appear to be proteins of body fluids. Circulating lipoproteins and skin matrix proteins emerge as potentially important health-related targets. Through oxidation of collagen, elastin and other proteins of the skin matrix, arNOXes are major contributors to skin aging through tyrosine and thiol oxidation and subsequent cross linking. The main destructive action of arNOX, however, may be to directly oxidize circulating lipoproteins. arNOX in the blood is structured as an integral component of the LDL particle through site-specific binding. As such, arNOXes are implicated as major risk factors for cardiovascular disease due to specific oxidation of LDLs. The superoxide produced and its conversion to hydrogen peroxide would be one part of the potentially destructive properties by contribution to lipid oxidation. Inhibition of arNOX proteins provides a rational basis for anti-aging interventions and their elimination as a major risk factor of atherogenesis.

Molecular cloning and characterization of an ECTO-NOX3 (ENOX3) of <i>Saccharomyces cerevisiae</i>

Exfoliated ECTO-NOX3 (ENOX3) proteins, are members of the human TM9 superfamily of transmembrane proteins that generate superoxide, are present in blood and other body fluids, and increase activity with age beginning about age 30, hence age-related NOX (arNOX or ENOX3). A yeast deletion library was screened based on NADH fluorescence using a 384 well plate assay to identify a yeast isolate lacking a previously identified cell surface oxidase exhibiting an oscillatory pattern with a period length of 26 min and capable of generating superoxide. The cDNA was cloned from a yeast over expression library using NADH as an impermeant substrate with analysis by Fast Fourier Transform and decomposition fits. The objective was to identify and sequence an ENOX homologue in Saccharomyces cerevisiae with a 26 min rather than a 24 or 25 min period length. The finding identified YER113C as the yeast ENOX3 protein with a 26 min period and capable of generating superoxide. The encoded protein was expressed in bacteria and characterized. Gel slices of expressed proteins revealed a protein of ca. 81,545 kDa with properties paralleling those of human ar-NOX (periodic NADH oxidation, protein disulfide thiol interchange, inhibited by mammalian arNOX inhibitors and superoxide production inhibited by superoxide dismutase). The YER113C sequence exhibited a 44% similarity and a 26% identity with the mammalian ENOX3 SF4 (arNOX SF4) of the TM9 superfamily of transmembrane proteins1. The YER113C deletion mutant lacked arNOX activity.