Analysis of Alpha-2 Macroglobulin from the Long-Lived and Cancer-Resistant Naked Mole-Rat and Human Plasma

Abstract

Background: The naked mole-rat (NMR) is a long-lived and cancer resistant species. Identification of potential anti-cancer and age related mechanisms is of great interest and makes this species eminent to investigate anti-cancer strategies and understand aging mechanisms. Since it is known that the NMR expresses higher liver mRNA-levels of alpha 2-macroglobulin than mice, nothing is known about its structure, functionality or expression level in the NMR compared to the human A2M.

Results: Here we show a comprehensive analysis of NMR- and human plasma-A2M, showing a different prediction in glycosylation of NMR-A2M, which results in a higher molecular weight compared to human A2M. Additionally, we found a higher concentration of A2M (8.3±0.44 mg/mL vs. and 4.4±0.20 mg/mL) and a lower total plasma protein content (38.7±1.79 mg/mL vs. 61.7±3.20 mg/mL) in NMR compared to human. NMR-A2M can be transformed by methylamine and trypsin resulting in a conformational change similar to human A2M. NMR-A2M is detectable by a polyclonal antibody against human A2M. Determination of tryptic and anti-tryptic activity of NMR and human plasma revealed a higher anti-tryptic activity of the NMR plasma. On the other hand, less proteolytic activity was found in NMR plasma compared to human plasma.

Conclusion: We found transformed NMR-A2M binding to its specific receptor LRP1. We could demonstrate lower protein expression of LRP1 in the NMR liver tissue compared to human but higher expression of A2M. This was accompanied by a higher EpCAM protein expression as central adhesion molecule in cancer progression. NMR-plasma was capable to increase the adhesion in human fibroblast in vitro most probably by increasing CD29 protein expression. This is the first report, demonstrating similarities as well as distinct differences between A2M in NMR and human plasma. This might be directly linked to the intriguing phenotype of the NMR and suggests that A2M might probably play an important role in anti-cancer and the anti-aging mechanisms in the NMR.

Fig 1. Phylogenetic tree of A2M.

Phylogenetic analysis of A2M protein sequences was done using the ClustalW2 onlinetool (https://www.ebi.ac.uk/Tool/phylogeny/clustalw2phylogeny). The compared A2M protein sequences were readout from the Uniprot database.

Fig 2. Plasma and liver protein analysis.

NMR, human plasma (30μg) and purified human A2M (2.5μg) were separated by native PAGE (4–20%) (A) and SDS-PAGE (4–20%) under non-reducing (B) and reducing conditions (C). The protein concentration of NMR and human plasma was determined according to Bradford (**—p<0.01) (D). Protein extracts from human and NMR liver (20 μg each) were separated by SDS-PAGE (4–20%) (E) and subjected to western blot analysis using a polyclonal rabbit anti-human A2M antibody (5 μg/mL) and a monoclonal mouse ß-subunit specific anti-human LRP1 antibody (10μg/mL) (F). (A2M –alpha-2 macroglobulin, Alb–albumin, IgG–immunoglobulin, Trf—transferrin).

Fig 3. RATE electrophoresis of NMR and human plasma and analysis of the receptor-binding properties of their A2M.

RATE electrophoresis was done using 50 μg native NMR and human plasma and 50 μg of methylamine and trypsin treated plasma, respectively. Isolated human A2M served as control. Methylamine as well as trypsin shift human and NMR-A2M from the slow- to the fast-moving form. To visualize the respective protein bands the gel was stained with Commassie (A). Purified LRP1 (100–500 ng) was spotted to a nitrocellulose membrane and incubated with either 10 μg/mL human or NMR plasma. BSA served as negative control. A2M binding to LRP1 was detected by a polyclonal rabbit anti-human A2M antibody (5 μg/mL) (B). To block the binding of A2M to LRP1 the spotted LRP1 was pre-incubated with 1.5 μM RAP for 30 min prior to the addition of human or NMR plasma (C). (☐ - native/slow form of A2M, ◆ –transformed/fast form of A2M).

Fig 4. Western blot of plasma from NMR and human for alpha-2 macroglobulin.

The determination of A2M in plasma samples was performed by electrophoresis in 7% SDS-gel using NMR and human plasma, 30 μg each, and purified human A2M (1–2.5 μg). A2M was quantified by Commassie staining using isolated human A2M to prepare a calibration curve (A). Western blot analyses using a native gradient PAGE (4–20%) was applied to detect the transformed form of A2M using the alpha-1 antibody (B) and a 7% non-reducing SDS-PAGE to detect A2M with a polyclonal antibody using 9 and 3 μg NMR or 3 μg human plasma and 250 ng isolated human A2M (C).

Fig 5. Anti-tryptic and proteolytic activity of NMR and human plasma.

The inactivation of 0.05 μg trypsin was titrated with increasing amounts of human (A) or NMR (B) plasma corresponding to 1–100 μg protein and measured by the conversion of BAPNA to p-nitroaniline at 405 nm. The intrinsic tryptic activity of NMR and human plasma was determined by measuring the conversion of BAPNA to p-nitroaniline induced by 1–100 μg plasma protein (C). (*—p<0.05)

Fig 6. Adhesion induction by NMR plasma supplementation in human fibroblasts.

The determination of the EpCAM protein in NMR and human liver samples were analysed by electrophoresis in 10% SDS-gel using 20 μg protein in conjunction with immunoblotting using a monoclonal mouse anti-human EpCAM antibody (A). Human fibroblasts were cultured in medium supplemented with 0.3 or 1% NMR plasma for 24h and, controls were supplemented with PBS (ctr) or 1% human (hu) plasma), respectively. Cells were treated by trypsin/EDTA solution for exactly 1 min, washed and the remaining adherent cells were stained by Gentiana solution. The absorbance of the released dye is directly proportional to the number of adherent cells (B) (**—p<0.01; *- 0.05). Western blot analysis for CD29, CD44, EpCAM and GAPDH was performed by electrophoresis in 8% SDS-gel using 10–25 μg protein after 0.3 or 1% NMR plasma supplementation for 24h. CD29, CD44, EpCAM and GAPDH were detected with respective monoclonal antibodies (C).