Objective. To identify a Golgi complex autoantigen bound by Sjogren's syndrome (SS) autoantibodies.Methods. Serum from a patient with secondary SS and anti-Golgi antibodies was used as a probe to isolate a complementary DNA (cDNA) insert from a HeLa cDNA library.Results. A 3.7-kb cDNA encoding a 56-kd recombinant protein was immunoprecipitated by the human anti-Golgi serum and immune rabbit serum. Western blot analysis showed that the immune rabbit sera recognized a protein of 97 kd (golgin-97), suggesting that the isolated clone contained a partial cDNA. The 5' upstream sequence was obtained by rapid amplification of the cDNA ends. The complete cDNA contained 4,860 basepairs, encoding a protein with a calculated M , of 88 kd. Antibodies to golgin-97 were found in 12 (20%) of 60 sera known to have anti-Golgi autoantibodies, and the majority of these sera (8 of 12, or 75%) were from patients who had secondary SS.Conclusion. Golgin-97 is a unique Golgi complex antigen that appears to be a target of SS autoantibodies.

The serum from a Sjö gren's syndrome patient with anti-Golgi antibodies was used as a probe to isolate a 4.6-kilobase pair cDNA insert from a HeLa cDNA library. Expression of the cDNA in Escherichia coli and the in vitro translation products of the cDNA yielded a recombinant protein that migrated in SDS-polyacrylamide gel electrophoresis at 180 kDa. This protein was immunoprecipitated by the human anti-Golgi serum and by immune rabbit serum but not by normal human serum or preimmune rabbit serum. Western blot analysis showed that the prototype human and immune rabbit sera recognized a 245-kDa protein, suggesting that the isolated clone contained a partial cDNA. The 5-upstream sequence obtained by the rapid amplification of cDNA ends methodology using human placental cDNA and the combined HeLa cDNA contained 6965 base pairs and encoded a protein of 245 kDa and, like other Golgi autoantigens described earlier, is highly rich in coiledcoils. The deduced amino acid sequence included the decapeptide ESLALEELEL, which was identified as one of two signature sequences previously reported in a family of peptide hormones and neuropeptides known as "granins." This is the first report of a Golgi complex autoantigen that bears structural similarities to the granin family of proteins.

Clostridium botulinum neurotoxin type A (BTx-A) is known to inhibit the release of acetylcholine at the neuromuscular junctions and synapses and to cause neuroparalysis and death. In this study, we have identified two monoclonal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal doses of BTx-A challenge. The neutralizing activities for BT57-1 and BT150-3 were 10 3 and 10 4 times the 50% lethal dose, respectively. Using immunoblotting analysis, BT57-1 was recognized as a light chain and BT150-3 was recognized as a heavy chain of BTx-A. Also, applying the phage display method, we investigated the antibodies' neutralizing B-cell epitopes. These immunopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-X-Asp-Cys for BT150. The synthetic peptide P4M (KGTFDPLQEPRT) corresponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically. This peptide was also shown by competitive inhibition assay to be able to inhibit phage clone binding to BT57-1. Aspartic acid (D 5 ) in P4M was crucial to the binding of P4M to BT57-1, since its binding activity dramatically decreased when it was changed to lysine (K 5 ). Finally, immunizing mice with the selected phage clones elicited a specific humoral response against BTx-A. These results suggest that phage-displayed random-peptide libraries are useful in identifying the neutralizing epitopes of monoclonal antibodies. In the future, the identification of the neutralizing epitopes of BTx-A may provide important information for the identification of the BTx-A receptor and the design of a BTx-A vaccine.

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