Characterisation of ACP5 missense mutations encoding tartrate-resistant acid phosphatase associated with spondyloenchondrodysplasia
| Autor: | Balakumar P. S. S. Murthy, Anthony J. Janckila, Janani Ramesh, Bhuvarahamurthy Venugopal, Ranga N. Parthasarathy, Latha K. Parthasarathy, Farhana Begum, Rif S. El-Mallakh, Ramya Murugan |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Metabolic Processes Glycosylation Mutant Biochemistry chemistry.chemical_compound 0302 clinical medicine Animal Cells Medicine and Health Sciences Enzyme-Linked Immunoassays Tartrate-resistant acid phosphatase chemistry.chemical_classification Multidisciplinary biology medicine.diagnostic_test Chemistry Transfection Proteases Recombinant Proteins Enzymes 030220 oncology & carcinogenesis Physical Sciences Medicine Cellular Types Research Article Missense Mutation Science Immune Cells Immunology Mutation Missense Antigen-Presenting Cells Research and Analysis Methods Osteochondrodysplasias Phosphates Autoimmune Diseases 03 medical and health sciences Western blot medicine Genetics Humans Immunoassays Tartrate-Resistant Acid Phosphatase Acid phosphatase Chemical Compounds Biology and Life Sciences Proteins Cell Biology Dendritic Cells Molecular biology 030104 developmental biology Enzyme Metabolism Amino Acid Substitution Cell culture Mutation Proteolysis biology.protein Enzymology Immunologic Techniques Mutant Proteins Serine Proteases |
| Zdroj: | PLoS ONE PLoS ONE, Vol 15, Iss 3, p e0230052 (2020) |
| ISSN: | 1932-6203 |
| Popis: | Biallelic mutations in ACP5, encoding tartrate-resistant acid phosphatase (TRACP), have recently been identified to cause the inherited immuno-osseous disorder, spondyloenchondrodysplasia (SPENCD). This study was undertaken to characterize the eight reported missense mutations in ACP5 associated with SPENCD on TRACP expression. ACP5 mutant genes were synthesized, transfected into human embryonic kidney (HEK-293) cells and stably expressing cell lines were established. TRACP expression was assessed by cytochemical and immuno-cytochemical staining with a panel of monoclonal antibodies. Analysis of wild (WT) type and eight mutant stable cell lines indicated that all mutants lacked stainable enzyme activity. All ACP5 mutant constructs were translated into intact proteins by HEK-293 cells. The mutant TRACP proteins displayed variable immune reactivity patterns, and all drastically reduced enzymatic activity, revealing that there is no gross inhibition of TRACP biosynthesis by the mutations. But they likely interfere with folding thereby impairing enzyme function. TRACP exists as two isoforms. TRACP 5a is a less active monomeric enzyme (35kD), with the intact loop peptide and TRACP 5b is proteolytically cleaved highly active enzyme encompassing two subunits (23 kD and 16 kD) held together by disulfide bonds. None of the mutant proteins were proteolytically processed into isoform 5b intracellularly, and only three mutants were secreted in significant amounts into the culture medium as intact isoform 5a-like proteins. Analysis of antibody reactivity patterns revealed that T89I and M264K mutant proteins retained some native conformation, whereas all others were in "denatured" or "unfolded" forms. Western blot analysis with intracellular and secreted TRACP proteins also revealed similar observations indicating that mutant T89I is amply secreted as inactive protein. All mutant proteins were attacked by Endo-H sensitive glycans and none could be activated by proteolytic cleavage in vitro. In conclusion, determining the structure-function relationship of the SPENCD mutations in TRACP will expand our understanding of basic mechanisms underlying immune responsiveness and its involvement in dysregulated bone metabolism. |
| Databáze: | OpenAIRE |
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