BETA-2-MICROGLOBULIN ISOFORMS IN HEALTHY-INDIVIDUALS AND IN AMYLOID DEPOSITS

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