Résumé
Beta 2 microglobulin isoforms in healthy individuals and in amyloid deposits. Beta 2 microglobulin (β2m) is classically known to have isoforms with isoelectric points (pI) 5.7 and 5.3. New isoforms of β2m 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 β2m. The other modifications in β2m 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 β2m 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 β2m content. Two-dimensional gel electrophoresis (2D-PAGE) of β2m 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 β2m antibody by Western blotting, confirming that they were β2m isoforms. β2m isoforms from the amyloid deposits were then separately purified with an IEF column (PB94, Pharmacia™) for analysis. Enough quantities of three pure β2m 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 β2m. In two of them β2m 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 β2m isoforms in gel isoelectrofocusing contain β2m with intact N-terminus. None of them have deamidated their 17th residue. More importantly, the β2m 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 β2m. They suggest that the precipitation of β2m into amyloid fibrils should result from the interaction of β2m with other factors with amyloid enhancing activity.