Chemical modification of insulin in amyloid fibrils

Abstract

We have investigated the chemical modification of insulin under conditions that promote the conversion of the soluble protein into amyloid fibrils. The modifications that are incorporated into the fibrils include deamidation of Asn A21, Asn B3, and Gln B4. In order to prepare fibrils with minimal deamidation of these residues, the kinetics of aggregation were accelerated by seeding with aliquots of a solution containing preformed fibrils. The resulting fibrils were then reincubated to determine the extent to which chemical modification occurs in the fibril itself. The deamidation of Asn A21 in particular could be followed in detail. Deamidation of this residue in the fibrillar form of insulin was found to occur in only 52 +/- 5% of molecules. This result indicates that there are at least two different packing environments of insulin molecules in the fibrils and suggests that the characterization of chemical modifications may be a useful probe of the environment of polypeptide chains within amyloid fibrils.

Figure 1.

Insulin sequence and site of modifications. Primary structure of insulin with the A-chain shown above and the B-chain shown below. The A8–A9 split product (boxed) and asparagine deamidation (circled N) have been observed in therapeutic insulin preparations (Brange et al. 1992). In this work, the modified forms of insulin identified in insulin amyloid fibrils include both deamidated asparagines (positions A21 and B3) and deamidation of glutamine at position B4 (circled Q).

Figure 2.

HPLC and IEF of solubilized insulin fibrils. (A) HPLC traces of solubilized insulin fibrils under nonreducing HPLC conditions reveal two peaks, I and I′. The peak labeled I corresponds to unmodified insulin and insulin with B-chain deamidation (determined by IEF). The I′ peak corresponds to insulin with A-chain deamidation and insulin with both A-chain deamidation and B-chain deamidation (IEF). The arrow indicates the expected relative retention time of the A8–A9 split product (Brange et al. 1992). The extent of Asn A21 deamidation can be approximated by the relative ratio of I′ to I. The Asn A21 deamidation in the 24 h unseeded fibrils is greater than the modification of the 8 h seeded + 16 h fibrils, indicating less chemical modification in the fibrils than in solution. (B) IEF gel electrophoresis of solubilized insulin amyloid fibrils formed by incubation for 24 h. IEF was performed under nonreducing (lane 1, OX) and reducing (lane 3, RED) conditions and compared with IEF marker proteins (lane 2) corresponding to pI 7.2, 6.4, 5.9, 5.1, 4.6, and 3.3 from top to bottom. Bands that occur in the same position as for an untreated insulin sample are labeled with I (insulin), A (A-chain), or B (B-chain) respectively. IEF under nonreducing conditions reveals insulin (calc. pI 5.4) and two additional bands. The dark band closest to insulin is assigned to insulin deamidated at position Asn A21 and the fainter bands to deamidation at Asn B3 (based on previous assignment by Brange et al. 1992) or insulin deamidated at more than one position (a single deamidation would reduce the pI by 0.4–0.5 and double deamidation by 0.7–0.8). Under reducing conditions, the B-chain (calc. pI 6.9) and a deamidated form of the B-chain (a single deamidation would reduce the pI by 0.9) are clearly distinguishable. These two bands were sequenced to reveal the presence of deamidated Asn B3 and Gln B4. The A-chain (both in control insulin solutions and in fibrils) smears on the gel, possibly due to reoxidation.

Figure 3.

Time dependence of the deamidation of Asn A21 in insulin fibrils. (A) Insulin fibrils formed by seeding (top) and then reincubated at 65°C (pH 2.0) for 3 or 13 d (middle and bottom) were analyzed by HPLC under reducing conditions. The A-chains and B-chains are clearly resolvable (A,B) and the deamidation that occurs can be detected (A′, B′). After 13 d of incubation, the relative amount of A-chains that are deamidated at Asn A21 (A′) is very close to 50%. (B) Time course for deamidation of Asn A21 in the fibrils. The percent of deamidated Asn A21 was determined from HPLC analysis (e.g., Fig. 3A ▶); squares and diamonds represent two different batches of insulin. The maximum amount of deamidated Asn A21 in the fibrils is 52 ± 5%.