The melibiose-derived glycation product mimics a unique epitope present in human and animal tissues

Abstract

Non-enzymatic modification of proteins by carbohydrates, known as glycation, leads to generation of advanced glycation end-products (AGEs). In our study we used in vitro generated AGEs to model glycation in vivo. We discovered in vivo analogs of unusual melibiose-adducts designated MAGEs (mel-derived AGEs) synthesized in vitro under anhydrous conditions with bovine serum albumin and myoglobin. Using nuclear magnetic resonance spectroscopy we have identified MAGEs as a set of isomers, with open-chain and cyclic structures, of the fructosamine moiety. We generated a mouse anti-MAGE monoclonal antibody and show for the first time that the native and previously undescribed analogous glycation product exists in living organisms and is naturally present in tissues of both invertebrates and vertebrates, including humans. We also report MAGE cross-reactive auto-antibodies in patients with diabetes. We anticipate our approach for modeling glycation in vivo will be a foundational methodology in cell biology. Further studies relevant to the discovery of MAGE may contribute to clarifying disease mechanisms and to the development of novel therapeutic options for diabetic complications, neuropathology, and cancer.

Figure 1

Autoantibodies present in human serum bind different model AGEs. (A) 12.5% SDS-PAGE separation of MB (lane 8) and its glycation products obtained in HTG reaction with: lac (lane 1), mal (lane 2), cel (lane 3), mel (lane 4), glc (lane 5), man (lane 6), gal (lane 7). Molecular mass is indicated with lines on the left side of the picture. (B) WB of serum from diabetic patient with control unmodified protein MB (lane 8), BSA (lane 9), and model AGEs: MB-lac (lane 1), MB-mal (lane 2), MB-cel (lane 3), MB-mel (lane 4), MB-glc (lane 5), MB-man (lane 6), MB-gal (lane 7), BSA-lac (lane 10) formed in HTG or HPG conditions (lane 1–7 and 10, respectively). (C) ELISA of serum from diabetic patients on a plate coated with unmodified MB (open circle) or MB glycated by different carbohydrates (filled marks); the control of secondary Ab were evaluated on wells with BSA as a blocking agent (open square); values presented are of serum from a representative patient; (D) ELISA with representative sera from diabetic (DM), diabetic nephropathy (NPH) and Buerger’s disease (BD) patients on plate coated with MB-mel; the results (A₁ 490) were normalized by subtraction of the absorption of secondary Ab (PBS in place of serum).

Figure 2

Fractionation of products obtained in HTG reaction of MB with mel (MAGEs fr. 1–3). (A) typical elution profile from a Sephadex G-200 column: tubes with eluted material were pooled as fractions 1–3 for analysis while low molecular mass material was ignored. (B) Products present in fraction 1, 2, and 3 (lane 1–3) and MB used for glycation (lane 4) were analyzed on 12.5% SDS-PAGE gel. The molecular mass of the reference standards is indicated on the left.

Figure 3

Characterization of the polyclonal anti-MAGE antisera. ELISA of anti-MB-mel, anti-BSA-mel, and anti-RIg-mel rabbit sera on plates coated with (A) MB-mel (solid lines) or MB (dashed lines); (B) BSA-mel (solid lines) or BSA (dashed lines); (C) RIg-mel (solid lines) or RIg (dashed lines); the results were normalized by subtraction of the absorption of secondary Ab (PBS in place of serum) and expressed as A₁ 490. The glycation products generated from melibiose show different antigenic properties than products formed from (D) glycolaldehyde (GA) and (E) methylglyoxal (MGO). The samples were as follows: (1) BSA-GA (24 µg), (1′) BSA-MGO (2 µg), (2) BSA-mel (1 µg), (3) BSA (2 µg) were resolved in 10% polyacrylamide gel stained with CBB (d1,e1) or transferred onto PVDF membrane for Western blotting (d2,e2) with the anti-MAGE (MB-mel) rabbit serum.

Figure 4

Reactivity of anti-MAGE mAb with the model glycation products. The MB (lane 16) was incubated in solution (lanes 2, 4, 6, 8, 11, 13) or in dry conditions (3, 5, 7, 9, 12, 14) with mel (2, 3), lac (4, 5), fru (6, 7), glc (8, 9), MGO (11, 12), GA (13, 14). The protein glycation products (50 µg/well) separated on 12.5% SDS-PAGE gel were stained with Coomassie Brilliant Blue (A) or transferred onto the membrane probed with the anti-MAGE/10 mAb (B).

Figure 5

Purification and characterization of the LMW MAGE generated from mel and Nα-acetyl-lysine (NAL). (A) Elution profile of the LMW MAGEs generated on the HW-40S column recorded at 225 nm; the eluted material was pooled resulting in fractions 1, 2, and 3; (B) Fraction 2 was tested in competitive ELISA on a plate coated with 1 µg/well MB-mel; 0–600 μg/ml LMW MAGEs fr.2 was used as a competitor; fluorescence profile within 370–500 nm after excitation at 350 nm was recorded for 1 mg/ml solutions of (C) NAL, mel, LMW MAGEs and (D) protein-bound AGEs (MB, MB-mel).

Figure 6

LC-TOF–MS analysis of the LMW MAGEs. (A) Mass spectrum of the major ion with m/z 513 and (B) its MS/MS fragmentation pattern.

Figure 7

NMR spectrum of the LMW product formed from melibiose and N-α-acetyl-lysine. The inset shows the solved structure of the novel MAGE product.

Figure 8

MAGE accumulation in human and animal muscles. Tissue sections from human (A), horse (B), pig (C), rabbit (D), rat (E), chicken (F), frog (G), fish (H), and snail (I) were stained with hematoxylin–eosin (H&E) in addition to the monoclonal antibodies anti-MAGE/10 (odd numbers) or with H&E only (even numbers). The representative pictures of human skeletal muscles (1, 2), horse skeletal muscles (3, 4), pig skeletal muscles (5, 6), pig heart (7, 8), pig intestinal smooth muscles (9, 10), rabbit adipose tissue (11, 12), rabbit connective tissue (13, 14), rat heart (15, 16), rat intestinal smooth muscles (17, 18), chicken heart (19, 20), frog skeletal muscles (21, 22), fish skeletal muscles (23, 24), snail muscle tissue (25, 26) are shown. Black arrows show positive staining of skeletal (1, 3, 5, 21, 23), heart (7, 15, 19) and smooth muscle (9, 17, 25) myocytes; black arrowhead shows negative staining of smooth muscle myocytes (25); blue arrows show positive staining of arterial myocytes (17); red arrows show positive staining of connective tissue (13, 25); white arrows show positive staining of adipose tissue (11). The scale bars show 50 μm.