Human ADA2 belongs to a new family of growth factors with adenosine deaminase activity

Abstract

Two distinct isoenzymes of ADA (adenosine deaminase), ADA1 and ADA2, have been found in humans. Inherited mutations in ADA1 result in SCID (severe combined immunodeficiency). This observation has led to extensive studies of the structure and function of this enzyme that have revealed an important role for it in lymphocyte activation. In contrast, the physiological role of ADA2 is unknown. ADA2 is found in negligible quantities in serum and may be produced by monocytes/macrophages. ADA2 activity in the serum is increased in various diseases in which monocyte/macrophage cells are activated. In the present study, we report that ADA2 is a heparin-binding protein. This allowed us to obtain a highly purified enzyme and to study its biochemistry. ADA2 was identified as a member of a new class of ADGFs (ADA-related growth factors), which is present in almost all organisms from flies to humans. Our results suggest that ADA2 may be active in sites of inflammation during hypoxia and in areas of tumour growth where the adenosine concentration is significantly elevated and the extracellular pH is acidic. Our finding that ADA2 co-purified and concentrated together with IgG in commercially available preparations offers an intriguing explanation for the observation that treatment with such preparations leads to non-specific immune-system stimulation.

Figure 1. Characterization of ADA from IgG preparations

(A) Typical elution profile showing separation of the substrates and products of three reactions of purine metabolism on a column with Toyopearl TSK HW-40 S gel. PNP, purine nucleotide phosphorylase; HX, hypoxanthine. A 400 μl mixture containing 5′-AMP, adenosine, inosine and hypoxanthine was injected into an XK 16/20 column packed with 10 ml of Toyopearl TSK HW-40 S gel and equilibrated with 0.01 M sodium phosphate buffer. The nucleotides and nucleosides, which are specifically retained by the gel matrix, were subsequently eluted in the same buffer. The ratio between the peak areas was used to calculate the rates of the corresponding enzymatic reactions. (B) ADA2 activity in ten commercial IgG preparations in comparison with the ADA2 activity in human plasma. ADA2 is co-purified and concentrated together with the immunoglobulin fraction during industrial processing of human plasma using cold ethanol precipitation. (C) pH dependence of ADA activity in IgG preparations. The acid optimum pH of the reaction is a characteristic feature of the ADA2 isoenzyme. (D) Determination of K_m value for ADA in IgG preparations using a Lineweaver–Burk plot.

Figure 2. Purification of ADA2 from IgG preparations

(A) Elution of ADA2 from heparin–Sepharose using a linear NaCl gradient. (B) ADA2 purification using a High Load Superdex 200 gel-filtration column. Inset: SDS/PAGE analysis of ADA2 after gel filtration. (C) A table summarizing the purification steps of ADA2.

Figure 3. ADA2 is encoded by the CECR1 gene

(A) Comparison of amino acid sequences of CECR1 protein (ADA2) from Homo sapiens (H. sap) (Entrez Protein database accession no. AAF65941) with a hypothetical protein from Gallus gallus (G. gal) (Entrez Protein database accession no. CAG31985) using the Stretcher program [41]. The peptides of ADA2, which were identical with the peptides derived from the CECR1 sequence, are highlighted (see Table 1 for comparison). Potential sites of N-glycosylation are shown in boldface. The signal sequences of the proteins predicted by SignalP 3.0 Server [42] are underlined. (B) Comparison of the amino acid sequences of the N-terminal region of ADA2 from G. gallus [20] and the amino acid sequence of a hypothetical protein from G. gallus (Entrez Protein database accession no. CAG31985). The signal sequence of G. gallus CECR1 is underlined.

Figure 4. Catalytic parameters and a possible physiological role of ADA2

(A) Determination of the catalytic parameters of purified ADA2 using a Lineweaver–Burk plot. (B) ADA2 expressed by activated macrophages might be involved in signal-transduction pathways via adenosine receptor A₃. M. t, M. tuberculosis.