Expression, characterization, processing and immunogenicity of an insulin-dependent diabetes mellitus autoantigen, IA-2, in Sf-9 cells

Abstract

Autoantibodies to a 64-kD protein and a 40-kD tryptic fragment from pancreatic islets have been detected at high frequency in the sera of patients with insulin-dependent diabetes mellitus (IDDM). IA-2, a newly isolated transmembrane protein tyrosine phosphatase, is a major islet cell autoantigen in IDDM and the precursor of a 40-kD tryptic fragment. To express large quantities of recombinant IA-2 protein and analyse post-translational modifications we expressed full-length human IA-2 in baculovirus-infected Sf-9 cells. IA-2 expression was analysed by Western blot and by immunoprecipitation of 35S-methionine-radiolabelled proteins with rabbit antisera or IDDM sera. A 120-kD IA-2 protein was detected during the early, but not the late, phase of the infection. Pulse-chase experiments showed that the 120-kD protein was processed into fragments of 64 kD and smaller fragments of approximately 50 kD, 38 kD and 32 kD. The 64-kD fragment appeared as a doublet. Tunicamycin and PNGase F treatment down-shifted the 120-kD protein and the 64-kD doublet into lower molecular weight bands, suggesting that both were glycosylated. Trypsin treatment converted the 120-kD protein and the 64-kD doublet into a 40-kD fragment. Baculovirus-expressed IA-2 was as sensitive or slightly more sensitive than in vitro translated IA-2 in detecting autoantibodies to IA-2: 66% of sera from newly diagnosed IDDM patients reacted with baculovirus-expressed IA-2 compared with 59% of the same sera which reacted with in vitro translated IA-2. It is concluded that baculovirus-expressed IA-2 is a good source of autoantigen and that a number of lower molecular weight fragments with which IDDM autoantibodies react are derived from the 120-kD full-length IA-2 molecule.

Fig. 1

Expression of human IA-2 protein in Bac-hIA-2-infected Sf-9 cells. Uninfected (a) and 2 day-infected (b) Sf-9 cells were stained with rabbit antisera to IA-2 intracellular domain.

Fig. 2

Time course of Bac-hIA-2 expression in Sf-9 cells. At 24, 48, 72 and 96 h post-infection cellular lysates were subjected to 10% SDS–PAGE and immunoblotted with rabbit antiserum to the intracellular domain of human IA-2. The arrows point to the 120-kD full-length human IA-2 protein and the 64-kD IA-2 doublet. The bracket shows degradation products of IA-2. Wild-type baculovirus (AcNPV).

Fig. 3

Molecular processing of Bac-hIA-2 protein in Sf-9 cells. Sf-9 cells were pulsed for 30 min with³⁵S-methionine (zero time) and chased for 1, 2, 4, 9 and 24 h. Cellular lysates were then immunoprecipitated with anti-IA-2⁺ insulin-dependent diabetes mellitus (IDDM) sera and subjected to 10% SDS–PAGE. Arrows point to the 120-kD and 64-kD IA-2 proteins and the various fragments of IA-2 being processed during the chase

Fig. 4

Effect of tunicamycin and peptide:N-glycosidase F (PNGase F) on Bac-hIA-2 protein. Sf-9 cells were treated with 0.1% DMSO or tunicamycin (10 μg/ml) before being infected with Bac-hIA-2 recombinant virus and labelled with ³⁵S-methionine. The cell lysates then were immunoprecipitated with insulin-dependent diabetes mellitus (IDDM) sera and subjected to 10% SDS–PAGE. Immunoprecipitated proteins were treated with PNGase F (lane 5) before being subjected to gel separation. The arrows show the molecular mass and down-shift of the 120- and 64-kD proteins.

Fig. 5

Trypsin treatment of Bac-hIA-2 protein. Infected Sf-9 cell lysates were radiolabelled with ³⁵S-methionine, immunoprecipitated with insulin-dependent diabetes mellitus (IDDM) sera, treated with trypsin (50 μg/ml) for 30 min, and then subjected to 10% SDS–PAGE. The 120-, 64- and 50-kD IA-2 proteins were converted into a 40-kD tryptic fragment.

Fig. 6

Immunoprecipitation of baculovirus-expressed human IA-2 with insulin-dependent diabetes mellitus (IDDM) serum. Sf-9 cells were labelled with ³⁵S-methionine 48 h after infection with Bac-hIA-2 recombinant virus. Various amounts of labelled cell lysates (lanes 1 and 4, 40 000 ct/min; lanes 2 and 5, 80 000 ct/min; and lanes 3 and 6, 60 000 ct/min) were immunoprecipitated either with IDDM sera (lanes 1–3) or control sera (lanes 4–6) and then subjected to 10% SDS–PAGE.

Fig. 7

Comparison of the reactivity of baculovirus-expressed IA-2 and in vitro translated IA-2 with insulin-dependent diabetes mellitus (IDDM) sera. Sera from 59 new-onset IDDM patients and 46 healthy controls were tested by radioimmunoprecipitation with either baculovirus-expressed IA-2 (80 000 ct/min) or in vitro translated IA-2 (100 000 ct/min).

Fig. 8

Correlation between the intensity of immunoprecipitated IA-2 bands with baculovirus-expressed IA-2 and in vitro translated IA-2 (r = 0.89, P < 0.0001).

Fig. 9

Post-translational modification of IA-2. Full-length IA-2 is processed by glycosylation and protease cleavage. The dibasic amino acids (KK) represent the potential cleavage sites from which the 64-kD doublet is generated. Trypsin treatment converts the 120-kD and 64-kD proteins into a 40-kD fragment.