caa a22 4500 606235949 CHVBK 20210128101245.0 cr unu---uuuuu 210128e20151201xx s 000 0 eng 10.1007/s11010-015-2534-8 doi (NATIONALLICENCE)springer-10.1007/s11010-015-2534-8 Identification and characterization of a bovine sperm acrosomal matrix protein and its mechanism of interaction with acrosomal hydrolases [Elektronische Daten] [Subir Nagdas, Linda Smith, Allen Mcnamara, Luisa Hernandez-Encarnacion, Ilza Medina-Ortiz] Fertilization, the union of male and female gametes to create offspring, is an intricate biological process dependent upon several biochemical and physiological events. Our understanding of the functions of protein constituents of the outer acrosomal membrane-associated matrix complex (OMC) is limited. A highly purified OMC fraction isolated from bovine cauda sperm heads comprised 54, 50, 45, and 38-19kDa polypeptides. The objective of this study is to identify and characterize the 45kDa (OMC45) polypeptide, to define its role in binding acrosomal hydrolases, and to examine the fate of OMC45 polypeptide during the acrosome reaction. We isolated OMC45 polypeptide from the high-pH insoluble fraction of OMC. Proteomic analysis of OMC45 by MALDI-TOF-TOF yielded eight peptides that matched the NCBI database sequence of Tektin 3 (TEKT3). Triton X-100-permeabilized cauda sperm exhibited intense staining of the acrosomal segment with anti-OMC45 and anti-TEKT3. The OMC45 polypeptide was solubilized by radio-immunoprecipitation assay buffer extraction. The solubilized fraction was subjected to immunoprecipitation analysis. The OMC45 polypeptide was recovered in the anti-OMC45 immunoprecipitation pellet. An identical blot stained with anti-TEKT3 exhibited the presence of TEKT3 polypeptide in the anti-OMC45 pellet. Our immunofluorescence and biochemical studies confirm the proteomics identification of OMC45 polypeptide and that it exhibits a sequence similarity to TEKT3. OMC45 glycoprotein possesses both N-linked and O-linked oligosaccharides. Deglycosylated OMC45 revealed a significant reduction in both acrosin and N-acetylglucosaminidase (NAGA) binding in comparison with acrosin and NAGA binding to a native OMC45 polypeptide, demonstrating the important role of oligosaccharides in hydrolase binding. OMC45 polypeptide is not released during the acrosome reaction but remains in the particulate cell subfraction, associated with the hybrid membrane complex. Springer Science+Business Media New York, 2015 Bovine sperm nationallicence Acrosome nationallicence Glycoproteins nationallicence Tektin 3 nationallicence Acrosomal hydrolases nationallicence Nagdas Subir Department of Chemistry and Physics, Fayetteville State University, 1200 Murchison Road, 28301, Fayetteville, NC, USA aut Smith Linda Department of Chemistry and Physics, Fayetteville State University, 1200 Murchison Road, 28301, Fayetteville, NC, USA aut Mcnamara Allen Department of Chemistry and Physics, Fayetteville State University, 1200 Murchison Road, 28301, Fayetteville, NC, USA aut Hernandez-Encarnacion Luisa Department of Chemistry and Physics, Fayetteville State University, 1200 Murchison Road, 28301, Fayetteville, NC, USA aut Medina-Ortiz Ilza Department of Chemistry and Physics, Fayetteville State University, 1200 Murchison Road, 28301, Fayetteville, NC, USA aut Molecular and Cellular Biochemistry Springer US; http://www.springer-ny.com 410/1-2(2015-12-01), 11-23 0300-8177 410:1-2<11 2015 410 11010 https://doi.org/10.1007/s11010-015-2534-8 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s11010-015-2534-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Nagdas Subir Department of Chemistry and Physics, Fayetteville State University, 1200 Murchison Road, 28301, Fayetteville, NC, USA aut NATIONALLICENCE 700 1- Smith Linda Department of Chemistry and Physics, Fayetteville State University, 1200 Murchison Road, 28301, Fayetteville, NC, USA aut NATIONALLICENCE 700 1- Mcnamara Allen Department of Chemistry and Physics, Fayetteville State University, 1200 Murchison Road, 28301, Fayetteville, NC, USA aut NATIONALLICENCE 700 1- Hernandez-Encarnacion Luisa Department of Chemistry and Physics, Fayetteville State University, 1200 Murchison Road, 28301, Fayetteville, NC, USA aut NATIONALLICENCE 700 1- Medina-Ortiz Ilza Department of Chemistry and Physics, Fayetteville State University, 1200 Murchison Road, 28301, Fayetteville, NC, USA aut NATIONALLICENCE 773 0- Molecular and Cellular Biochemistry Springer US; http://www.springer-ny.com 410/1-2(2015-12-01), 11-23 0300-8177 410:1-2<11 2015 410 11010