| Myasthenia gravis(MG) is an antibody-mediated autoimmune disease of the neuromuscular junction[1,2]. Auto-antibodies to the muscle nicotinic acetylcholine receptor (AChR) is widely accepted as the main pathogenic agents[3]. While about 15% MG patients with typical generalized weakness have no detectable AChR antibodies, which known as seronegtive MG(SNMG)[4,5] can't be explained by AchR antibodies. These patients have a good response to immune therapy, and MG symptoms could be passively transferred to animals through injection sera of these patients'[5-7]. These results show that there were some other autoantigens that contribute to the pathogenesis of MG. In the past 40 years, several other antibodies have been found to be associated with MG, such as antibodies to a striated muscle antigen titin mainly present in MG patients with thymoma (MGT)[8]; Antibody to ryanodine receptor (RyR) related to late-onset MG and MGT[9]; Antibodies to pre-synaptic membrane receptor (PrsmR) can be detected in two third patients with MG([10-11]). And antibodies to a newly identified receptor tyrosine kinase MuSK(muscle specific kinase) present in 0-70% SNMG patients[12]. These patients often have bulbar involvement and reduced response to conventional immunosuppressive treatments[13], which may indicate another distinct form of disease[14]. Some cytokines such as TNF, IL-15[15,16] also have been reported to be changed in MG patients. Furthermore, Genetic susceptibility could also explain some MG patients because HLA phenotypes[17], AChR subgroups , Immunoglobulin heavy chain gene[19] have been reported to be associate to MG. It seems that MG pathogenesis is more complex rather than circulating antibodies to the AChR alone. The heterogeneous mechanism of MG has been widely accepted. Most studies have been focused on immuno-pathogenesis of MG by now. In recent years, our department have attempted to analyze the component changes in skeletal muscles. On the gene level, we searched for different expression genes between MG patients and healthy controls using Differential Display PCR(DD-PCR)[21]. Five dif-ferential expression genes were found. Among these 5 genes, p9 gene which is over-expressed in MG patients was identified as a new gene and has been partially cloned[22]. On the protein level, Ren et al[23>24] analyzed muscle extracts from MG patients, normal controls and other muscular disease(OMD) with SDS-PAGE electro-phoresis and stained with coomassie brilliant blue. His results showed that the expression of a protein with the molecular weight of 25000 dalton (P25 for abbreviation) was markedly decreased in the skeletal muscles of MG patients. While, since coomassie blue stain could not specifically reflect the level of a single protein, the specific decrease of P25 in MG patients needs further confirmation. P25 could be easily extracted with Guba-straub solution or 0.3M PBS without any detergent, which showed that it is a soluble protein in physiological conditions. Further analysis showed that it is a glycoprotein and is not any component of AChR group1251. While, what is it and how about its distribution and localization in skeletal muscles? To answer these questions, we expanded our study as follows.Part 1: Comparation of P25 levels in skeletal muscles from different patients groupsP25 protein was extracted and purified by using praparitive electrophoresis, and anti-sera against P25 protein were produced by immunizing BALB/c mice. The titers and specificity of antibodies were determined by ELISA and immuno-Western blotting respectively. The result of ELISA showed that the titer of the antisera was 1:3200. Immunoblot showed a single band corresponding to the molecular weight of 25kD in crude skeletal muscle extract which infer that antibodies to P25 have good specificity. We detected the level of P25 in skeletal muscles from 24 normal subjects, 28 MG and 10 ONMD patients. The results showed that Relative density values of MG patients were 1.04±0.18, and that of normal subjects and ONMD patients were 1.27±0.21 and 1.21 ±0.15 respectively. Statistic analysis showed that the expression of P25 in MG patients were significantly lower than that of normal subjects (PO.001) and OMD patients(P=0.012), but there was no significant difference between normal controls and OMD patients(P=0.431). Among subgroups of MG patients, differences betweengroup of MGT and MGH (P=0.502) and group of AChR positive and negative (P=0.996) have no statistic significance. The density values decreased with the clinical severity, but there was no statistic difference between these groups (P>0.1).Part 2: Tissue distribution and localization of P25 protein in skeletal musclesTo detect the tissue and species distribution of P25 protein, 8 kinds of tissues from adult human and embryo and skeletal muscles from 8 species of animals were extracted and immunobloting was performed. The results showed that the expression of P25 is not ubiquitous. Among 8 kinds of adult and embryonic human tissues including skeletal muscle, smooth muscle, myocardium, thymus, brain, lung, kidney, skin, the P25 protein band could be detected only in skeletal muscles. Among 8 species of animals including bovine, swine, dog, rabbit, rat, mouse, chick and frog, only skeletal muscle extracted from swine could cross-react with ant-sera of P25.To further elucidate the localization of P25 protein in skeletal muscles, immuno-histochemistry and immunoelectron microscopy were performed. Result from immu-nohistochemistry showed that positive stains distributed linearly and discontinuously under the muscle membranes and diffusely in the cytoplasm as well. But in some muscle cells the immunostaining was very low and even no staining. Immunostaining of muscle from MG patient was much lower than that of normal control. Under the electron microscopy, immunostaining could be seen under the sarcolemma and no immunoreaction was observed in the cytoplasm, neither in the neucei and mitochondria. These results suggested that the ultrastructural localization of P25 is under the sarcolemma of skeletal muscle.Part 3: Sequence identification of P25 protein by using peptide mass fingerprintTo disclose the nature of P25 protein, P25 protein was purified through two dimen-tional electrophoresis to prepare samples for N-terminal partial amino acid sequencing. The result showed that P25 is N-terminal blocked and could not be sequenced through Edman Degradation.To further elucidate the nature of P25 protein, Peptide mass printing (PMF) ofP25 protein was aquired through matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) technique [26]. The ProteinProspector V 4.0.5 MS-FIT program*271 (ProteinProspector. UCSF. http://prospector.ucsf.edu) was used to search the NIH database of American (NCBInr.l0.15.2003). Parameters were: species= homo sapiens, PI range=3 to 10, peptide mass range=15kDa to 80kDa, maximum erro tolerance=0.5Da, Minimum Matches^ 4, a maximum of one missing tryptic cleavage. The result showed that three proteins with the registration number of 4885099, 115462 and 11641267 could match with P25 protein. 11641267 is a sperm specific protein and could be excluded due to its specific expression. 4885099 and 115462 are the same protein-skeletal muscle specific carbonic anhydrase III(CA III) l28]. This protein is similar to P25 protein on many aspects such as tissue content, tissue specific distribution, cellular localization, isoelectric point, N-terminus blockage'29'301 etc. Further analysis showed that the exact molecular weight of P25 protein is identical to that of CAIII and monoclonal antibody of CAIII could specifically react to P25 protein in the purified and mixture of muscle extract state. Finally, we can conclude that P25 protein is CAIII.In this study, we found that P25 protein is specifically insufficient in MG patients and it is a skeletal muscle specific cytosolic protein. P25 was identified as carbonic anhydrase III based on the results of peptide mass finger printing (PMF). CAIII is the third mumber of carbonic anhydrase super family. Members of this family can catalyze the interconversion of CO2 and HCO3"1311. CAIII is highly expressed in the skeletal muscle of mammals as well the liver of male rat and adipocytes of rodent'32'331. But in human, it is only expressed in skeletal muscles and mainly in type I muscle subtype'341. The function of CAIII including carbonic anhydrase activity, esterase activity and phosphatase activity'351. The physiological role of CAIII is to catalyze the transform of HCO3 to CO2 which is extremely permeable across the muscle cell membrane, and helps to regulate intracellular pH[36]. In this study, we found that CAIII is specifically insufficient in the skeletal muscle of MG patients. It is an important discovery to the research of MG as well as that of CAIII. The easily fatigability of... |
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