過去本實驗室觀察到一位類肉瘤症(Sarcoidosis)病人的抗核抗體檢測，呈現nuclear dot pattern，將病人血清與大白鼠肝細取物反應，會辨識58KDa蛋白。58KDa 蛋白經胺基酸定序，結果為 Calreticulin(CRT)。CRT，是一種鈣離子結合蛋白，主要存在細胞內質網，其蛋白表現量超過細胞內全部蛋白的 0.1﹪(除紅血球細胞)。CRT功能相當廣泛，除了與鈣離子結合外還扮演chaperone 的角色幫助新生蛋白進行醣化、訊號傳遞、細胞附著(cell adhesion)、吞噬及亦為一種自體抗原。 N 端 CRT(1~180A.a)稱vasostatin，是CRT的epitope部份，同時也會抑制腫瘤生長。因此我們利用cloning方式，取得CRT及vasostatin之 cDNA，進而蛋白表現，以探討vasostatin與Sarcoidosis和自體免疫疾病間之關係。我們利用ELISA及Western blot分析Sarcoidosis血清中之CRT及vasostatin抗原皆呈陰性，因此推測 nuclear dot並非由CRT抗體所造成。另外，亦利用ELISA分析 123位自體免疫風濕疾病患者中vasostatin抗體的表現，結果發現有 12 位(9.7﹪)是具有抗vasostatin自體抗體，其中70位RA患者有 9 位(12﹪)、32 位SLE 有1位(3﹪)、21位Sj gren's syndrome 有 2 位(9.5﹪)呈現陽性反應。綜合以上的結果我們發現抗CRT自體抗體與自體免疫疾病是有相關，同時與文獻指出抗CRT抗體會非專一性的出現在各種疾病是一致的。 Our previous study had shown that serum from a patient with sarcoidosis had nuclear dot staining pattern detected by indirect immunofluoresence (IIF). In immunoblot -ting the sera recognized a 58KDa protein using purified rat extracts as an antigen. The protein was later identified as Calreticulin (CRT). CRT is found predominantly in the endoplasmic reticulum of most eukaryotic cells, but is also present in other intracellular locations in different cell types, including the nucleus, cytoplasm, cytoplasmic granules and the cytoplasm side of the plasma membrane. These comprise functions of CRT with the lumen of the ER (chaperone and Ca+2 storage and signaling) and CRT-dependent modulation of cellular functions at the extra-ER sites (cell adhesion and gene expression). CRT is a human autoantigen that is intimately associated with autoantigenic components of the Ro/SSA ribonucleoprotein (RNP) complex. Vasostatin, the amino terminal protein of the CRT molecule, can inhibit angiogenesis and suppress tumor growth. In this study, recombinant CRT and vasostatin protein were purified from Escherichia coli and were used as substrate antigens in ELISA for screening anti-vasostatin antibodies in patients with sarcoidosis and various autoimmune diseases. Anti-vasostatin autoantibody was detected in 12 (9.7%) of 123 different autoimmune rheumatic diseases and three (15%) of 52 with cytoplasmic positive of ANA pattern. These sera with anti-vasostatin autoantibody included nine patients (12%) with RA, one (3%) with SLE, two (9.5%) with Sjgren's syndrome. We also demonstrated that CRT was not a protein of the nuclear dots in sarcoidosis by Western blot and ELISA. In conclusion, the CRT peptide analogus reacted in ELISA with sera from patients with SLE, SS, RA, which in agreement with previous observations, suggesting that anti-CRT autoantibodies are not restricted to any subset of the diseases.
