Beta 2 microglobulin isoforms in healthy individuals and in amyloid deposits

Abstract

beta 2 microglobulin (beta 2m) is classically known to have isoforms with isoelectric points (pI) 5.7 and 5.3. New isoforms of beta 2m with lower pI, probably due to modifications with advanced glycation end products, were found in the amyloid deposits of dialysis related amyloidosis (DRA), and they were proposed as the amyloidogenic forms of beta 2m. The other modifications in beta 2m from amyloid deposits are partial proteolysis and single amino acid replacement (Asn by ASp at position 17). However, there are no data on the sequence of the different isoforms of beta 2 m from amyloid deposits. Amyloid deposits surgically obtained from the carpal tunnel from 13 dialysis treated patients and urine from 10 healthy volunteers and 5 living-related kidney donors were analyzed for beta 2m content. Two-dimensional gel electrophoresis (2D-PAGE) of beta 2m from amyloid deposits showed the presence of four or more isoforms with pIs < 5.7. All the spots migrating at 12 kDa Mr region and between 4 and 6 pH reacted with rabbit anti-human beta 2m antibody by Western blotting, confirming that they were beta 2m isoforms. beta 2m isoforms from the amyloid deposits were then separately purified with an IEF column (PB94, Pharmacia) for analysis. Enough quantities of three pure beta 2m isoforms could be obtained in two cases. The sequence analysis showed an intact N-terminus in all the isoforms. There was Asn in the 17th residue in all the isoforms sequenced. 2D-PAGE of urine from 8 out of the 10 healthy volunteers showed the presence of beta 2m. In two of them beta 2m also displayed four different isoforms. At least four isoforms were observed in urine of all the kidney donors. The present study shows that the elution peaks of three different beta 2m isoforms in gel isoelectrofocusing contain beta 2m with intact N-terminus. None of them have deamidated their 17th residue. More importantly, the beta 2m isoforms with lower pI are not specific for amyloidosis as they were found in urine from kidney donors and in normal volunteers. These results bring into question the hypothesis that dialysis related amyloidosis is due to the known modifications on beta 2m. They suggest that the precipitation of beta 2m into amyloid fibrils should result from the interaction of beta 2m with other factors with amyloid enhancing activity.

Use of Anti‐(β2 Microglobulin) mAb to Study Formation of Amyloid Fibrils

“…The presence of two bands in the western blot of native electrophoresis (Fig. 1 a) demonstrates that the antibodies recognize also the acidic form of m-microglobulin [20]. The intensity of the immunostaining of native mmicroglobulin with the three mAbs showed a certain variability, which could be caused by the slightly higher affinity of mAb 14H3 with respect to 1F11 and 7B6 or to a different exposure of the corresponding epitope when the protein is bound to the Immobilon membrane.…”

Gastrointestinal β2microglobulin amyloidosis in hemodialysis patients: biochemical analysis of amyloid proteins in small formalin-fixed paraffin-embedded tissue specimens

“…Recently, we reported a new simple formic acid microextraction technique allowing the recovery and chemical typing of amyloid in small formalin-fixed biopsy specimens. 15,16 Application of this microtechnique in the present study, together with our small-scale amyloid purification method, 22 made possible the first biochemical characterization of the intestinal amyloid deposits in dialysis-related amyloidosis. Our study was based on previous findings 39,40 showing that amyloid, in contrast to many other proteins, is not affected by formalin fixation, probably because beta-pleated sheet conformation of amyloid prevents formalin infiltration.…”

“…33 The immunohistochemical amyloid typing methods are used routinely to provide this information, but in some instances, the obtained results can be inconclusive, especially in relation to AL proteins. 35 In this respect, our biochemical micromethods 15,16 applied in this study were helpful in identifying unequivocally the chemical nature of the deposited amyloid. (Figures 7 and 8).…”

“…[10][11][12] The appearance of these isoforms was explained by deamination of Asn17 13,14 or modification by advanced glycation end products (AGE), 10,12 but these findings have not been confirmed by other authors. 15 In addition, presence of acidic b 2 M isoforms has been demonstrated in normal urine, thus bringing up the question whether these isoforms are specific to dialysis-related amyloidosis. 15 The importance of the b 2 M structure-unrelated factors in the pathogenesis of dialysis-related amyloidosis has been proposed by implying the role of other proteins and cells under chronic inflammatory stress conditions and by raising questions of bioincompatibility of membranes or insufficient purity of water used for dialysis.…”

“…15 In addition, presence of acidic b 2 M isoforms has been demonstrated in normal urine, thus bringing up the question whether these isoforms are specific to dialysis-related amyloidosis. 15 The importance of the b 2 M structure-unrelated factors in the pathogenesis of dialysis-related amyloidosis has been proposed by implying the role of other proteins and cells under chronic inflammatory stress conditions and by raising questions of bioincompatibility of membranes or insufficient purity of water used for dialysis. 16,17 Thus, the existence of the diseasespecific modifications of b 2 M structure and their role in the pathogenesis of amyloid disease remain questionable.…”