An insight into the changes in human plasma proteome on adaptation to hypobaric hypoxia

Abstract

Adaptation to hypobaric hypoxia is required by animals and human in several physiological and pathological situations. Hypobaric hypoxia is a pathophysiological condition triggering redox status disturbances of cell organization leading, via oxidative stress, to proteins, lipids, and DNA damage. Identifying the molecular variables playing key roles in this process would be of paramount importance to shed light on the mechanisms known to counteract the negative effects of oxygen lack. To obtain a molecular signature, changes in the plasma proteome were studied by using proteomic approach. To enrich the low-abundance proteins in human plasma, two highly abundant proteins, albumin and IgG, were first removed. By comparing the plasma proteins of high altitude natives with those of a normal control group, several proteins with a significant alteration were found. The up-regulated proteins were identified as vitamin D-binding protein, hemopexin, alpha-1-antitrypsin, haptoglobin β-chain, apolipoprotein A1, transthyretin and hemoglobin beta chain. The down-regulated proteins were transferrin, complement C3, serum amyloid, complement component 4A and plasma retinol binding protein. Among these proteins, the alterations of transthyretin and transferrin were further confirmed by ELISA and Western blotting analysis. Since all the up- and down- regulated proteins identified above are well-known inflammation inhibitors and play a positive anti-inflammatory role, these results show that there is some adaptive mechanism that sustains the inflammation balance in high altitude natives exposed to hypobaric hypoxia.

Figure 1. Representative SDS-PAGE image of depleted human plasma sample from control and high altitude native (HAN).

Lane 1 and 3 represent crude plasma samples of control and HAN, respectively. Lane 2 and 4 represent depleted plasma samples of control and HAN, respectively. Lane M represents molecular weight marker.

Figure 2. Representative gel image of plasma proteins from HAN.

The proteins were resolved according to their isoelectric point (pI) in the first (5–8 pH) and their Mw on 12% SDS-PAGE followed by silver staining. Numbers mark protein spots were differentially expressed.

Figure 3. Gene ontology annotations of the proteins identified by MALDI-TOF/MS.

Results were obtained using Toppgene Suite. The distribution of identified proteins according to their (A) biological processes (B) molecular functions and (C) cellular functions.

Figure 4. Magnified comparison maps of spot (A) 25, (B) spot 8 and 9 in the 2-DE patterns of control and HAN.

Spot 25 had low expression in the control group but its expression increased in HAN. Spot 8 and 9 had high expression in the control group, but its expression decreased steadily in HAN.

Figure 5. ELISA analysis of controls and high altitude natives.

ELISA confirmed that the mean plasma concentration of transthyretin (A) was significantly increased and transferrin (B) was significantly decreased as compared to sea level controls (p<0.01). Data represents the Mean ± SD of three independent experiments. ‘*’ showed p<0.01 when compared to controls.

Figure 6. Western blot analysis of transthyretin and transferrin from plasma of controls and high altitude natives.

(A) Blots of transthyretin and transferrin were represented along with (B) their respective relative optical densities (ROD). Data represents the Mean ± SD of three independent experiments. Densitometry analysis of results from western blot, indicating significant change between the two groups compared by Student’s t-test. ‘*’ showed significantly different (p<0.05) when compared to controls.