226. Characterisation of a putative mouse sperm - zona pellucida receptor complex

2008 ◽  
Vol 20 (9) ◽  
pp. 26
Author(s):  
M. D. Dun ◽  
B. Nixon ◽  
R. J. Aitken
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Zona Pellucida ◽  
Molecular Chaperone ◽  
Reproductive Tract ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Female Reproductive Tract ◽  
Receptor Complex ◽  
High Molecular Weight Complex

Mammalian spermatozoa acquire the ability to fertilise an oocyte as they ascend the female reproductive tract. This process is characterised by a complex cascade of biophysical and biochemical changes collectively known as capacitation. The attainment of a capacitated state is accompanied by a dramatic reorganisation of the surface architecture which renders the spermatozoa competent to recognise the heterogenetic matrix of the zona pellucida surrounding the oocyte and initiate fertilisation. Emerging evidence from our laboratory indicates that this process is facilitated by molecular chaperone-mediated assembly of a multimeric receptor complex on the sperm surface. However, to date the presence and composition of such a complex has yet to be described. Through the novel application of blue native polyacrylamide gel electrophoresis (BN-PAGE), we have provided the first evidence that capacitated mouse spermatozoa express high molecular weight, multimeric protein complexes on their surface. Interestingly, at least two of these complexes contain heat shock protein 1 (HSPD1), a molecular chaperone that has previously been implicated in sperm-zona pellucida interaction. Furthermore, we were able to demonstrate that one of these complexes also possessed an affinity for solubilised zona pellucida as determined by Far-western blotting. 2D BN-PAGE was employed to further delineate the individual constituents of this high molecular weight complex, with several other chaperonin proteins not previously reported in functional sperm indentified. Collectively, these results support the notion the sperm-zona pellucida interaction are mediated by a multimeric receptor complex. Our current work is focussed on the identification of the key zona adhesion molecules that comprise this complex.

119. IDENTIFICATION AND CHARACTERISATION OF SURFACE PROTEIN COMPLEXES IN HUMAN SPERMATOZOA

2010 ◽  
Vol 22 (9) ◽  
pp. 37 ◽  
Author(s):  
K. A. Redgrove ◽  
B. Nixon ◽  
E. A. McLaughlin ◽  
M. K. O'Bryan ◽  
R. J. Aitken
Keyword(s):  
Zona Pellucida ◽  
Reproductive Tract ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Sperm ◽  
Female Reproductive Tract ◽  
Human Spermatozoa ◽  
Apical Region ◽  
Post Translational Modification ◽  
Cellular Mechanisms

A unique characteristic of mammalian spermatozoa is that upon ejaculation, they are unable to recognise and bind to an ovulated oocyte. These functional attributes are only realised following the sperms ascent of the female reproductive tract whereupon they undergo a myriad of biochemical and biophysical changes collectively referred to as ‘capacitation’. Since spermatozoa are both transcriptionally and translationally quiescent cells, this functional transformation must be engineered by a combination of post-translational modification and spatial reorganisation of existing sperm proteins. Indeed, evidence from our laboratory suggests that a key attribute of capacitation is the remodeling of the sperm surface architecture leading to the assembly and / or presentation of multimeric sperm-oocyte receptor complex(es). Through the novel application of Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE), we have secured the first direct evidence that human spermatozoa express a number of these protein complexes on their surface. Furthermore, we have demonstrated that a subset of these complexes harbour putative zona adhesion proteins and display strong affinity for solubilised zona pellucidae. In this study, we have extended our findings through the characterisation of one such complex containing arylsulfatase A (ASA), a protein with recognised affinity for sulfated ligands present within the zona pellucida. Through the application of immunohistochemistry and flow cytometry we revealed that ASA undergoes a capacitation-associated translocation to become expressed on the apical region of the human sperm head, a location compatible with a role in the mediation of sperm-zona pellucida interactions. This dramatic relocation was completely abolished by incubation of capacitating spermatozoa in exogenous cholesterol, suggesting that it may be driven in part by alteration in the membrane fluidity characteristics. Our current research is focused on confirming the role of ASA in human sperm-zona pellucida adhesion and elucidating the precise cellular mechanisms that underpin the proteins translocation to the cell surface.

168. IDENTIFICATION AND CHARACTERISATION OF SURFACE PROTEIN COMPLEXES IN HUMAN SPERMATOZOA

2009 ◽  
Vol 21 (9) ◽  
pp. 86
Author(s):  
K. A. Redgrove ◽  
E. A. McLaughlin ◽  
M. K. O'Bryan ◽  
R. J. Aitken ◽  
B. Nixon
Keyword(s):  
Zona Pellucida ◽  
Reproductive Tract ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Sperm ◽  
Female Reproductive Tract ◽  
Human Spermatozoa ◽  
20S Proteasome ◽  
Sperm Surface ◽  
Final Maturation

Upon leaving the testis mammalian spermatozoa are functionally incompetent and are thus unable to fertilize an oocyte. As the spermatozoa ascend the female reproductive tract, functional maturity is achieved through a complex cascade of biophysical and biochemical changes known as capacitation. An important aspect of this final maturation phase is the remodelling of the sperm surface architecture to enable it to interact with the zona pellucida, a glycoprotein matrix that surrounds the oocyte, and initiate fertilisation. While originally thought to be underpinned by a simple lock and key mechanism, emerging evidence has suggested that this interaction may instead be mediated by a multimeric recognition complex that is formed on the sperm surface during capacitation. However, to date the presence and composition of such a complex has yet to be described. Through the application of Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE), we have provided evidence that human spermatozoa express a number of high molecular weight protein complexes on their surface. Furthermore, the affinity of these surface expressed complexes for the zona pellucida was assessed utilising solubilised human zona pellucida and the technique of Far Western Blotting. Among the complexes that showed affinity for the zona pellucida we identified one comprising 14 subunits of the 20S proteasome. Interestingly, the 20S proteasome has previously been implicated in various aspects of mammalian fertilisation, including zona pellucida penetration and the acrosome reaction, although its precise role in these events has yet to be elucidated. Collectively, these results demonstrate the presence of multimeric protein complexes on the surface of human spermatozoa, and support their putative role in the initial interaction between the sperm and the zona pellucida. Our current research is focused on elucidation of the role of the 20S proteasome in human sperm-zona binding and further investigation of surface expressed protein complexes.

178. THE CHAPERONIN CONTAINING TCP-1 (CCT/TRiC) MULTISUBUNIT COMPLEX IS INVOLVED IN MEDIATING SPERM - OOCYTE INTERACTIONS

2010 ◽  
Vol 22 (9) ◽  
pp. 96
Author(s):  
M. D. Dun ◽  
R. Aitken ◽  
B. Nixon
Keyword(s):  
Zona Pellucida ◽  
Reproductive Tract ◽  
Protein Complexes ◽  
Female Reproductive Tract ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Sperm Surface ◽  
Collective Data ◽  
Concomitant Reduction ◽  
Future Work

Mammalian spermatozoa only express their capacity for fertilization following capacitation, a process characterized by a suite of biophysical and biochemical changes that occurs as the cells ascend the female reproductive tract. A key event associated with the attainment of a capacitated state is a dramatic reorganization of the sperm surface architecture to render these cells competent to bind to the protective matrix of the oocyte, the zona pellucida. Our previous analysis of these remodeling events has provided compelling evidence that they include the assembly and/or presentation of multimeric protein complexes on the sperm surface. In addition, we have demonstrated that at least two of these complexes possess strong affinity for solubilized zona pellucida. In our current study we have utilised mass spectrometry analysis to reveal that one of these complexes comprises the eight subunits that form a composite, multimeric structure known as the chaperonin containing TCP-1 (CCT/TRiC) complex. Our collective data suggest that this complex participates indirectly in zona pellucida interaction, possibly through the conveyance of key zona adhesion molecules to the sperm surface during capacitation. Consistent with this notion, we were able to demonstrate that the sperm CCT/TRiC complex releases its bound substrates upon exposure to ATP, and this treatment induced a significant, concomitant reduction in the ability of capacitated sperm to bind to the zona pellucida. Furthermore, the use of immunoprecipitation assays confirmed the interaction of the CCT/TRiC complex with at least one putative zona pellucida receptor candidate, namely zona pellucida binding protein 2 (ZPBP2). Future work is now aimed at identifying additional zona receptors that may reside within this complex and the pathways that regulate its functional assembly.

Factor VIII Is A Calcium-Binding Protein

1981 ◽  
Author(s):  
W H Cruickshank ◽  
E S Tackaberry ◽  
D S Palmer ◽  
G A Rock
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
High Molecular Weight ◽  
Column Chromatography ◽  
Calcium Binding ◽  
Essential Role ◽  
Polyacrylamide Gel Electrophoresis ◽  
Basic Structure ◽  
Low Molecular Weight ◽  
High Molecular Weight Complex

The high molecular weight complex of Factor VIII is known to dissociate into two subunits when chromatographed in a high ionic strength solution containing either calcium or sodium salts. Calcium also has an essential role in maintaining Factor VIII activity during storage (AABB 1980). These two findings suggest ah integral involvement of calcium in the basic structure-function relationship of the Factor VIII molecule. Consequently, we have examined the binding of 45Ca to both the low molecular weight (LMW) procoagulant subunit of Factor VIII and the high molecular weight (HMW) complex using PAGE and column chromatography on Sepharose 4B. When the HMW complex of Factor VIII was isolated from cryoprecipitate by standard chromatographic procedures, incubated with 45CaCl2 and subjected to polyacrylamide gel electrophoresis, the resultant autoradiogram demonstrated that all of the 45Ca was associated with the HMW material which did not enter the gel. When this HMW Factor VIII was incubated with 45Ca and then dissociated by column chromatography in a buffer containing 0.25 M CaCl2, all of the 45Ca was associated with the material eluting in the 2.3 Vo region. This corresponds to the elution volume of the LMW, procoagulant subunit of Factor VIII. Dialysis against buffer was not effective in removing the 45Ca. The data indicate that calcium is tightly associated with the low molecular weight subunit of Factor VIII and presents further corroborative evidence that calcium has an essential role in determining both the chemical and physical properties of the Factor VIII molecule.

Evolutionary insights into the microneme-secreted, chitinase-containing high molecular weight protein complexes involved in Plasmodium invasion of the mosquito midgut

2021 ◽  
Author(s):  
Hargobinder Kaur ◽  
M. Andreina Pacheco ◽  
Laine Garber ◽  
Ananias A. Escalante ◽  
Joseph M. Vinetz
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Protein Complexes ◽  
Peritrophic Matrix ◽  
High Molecular Weight Complex ◽  
Mosquito Midgut ◽  
Weight Protein ◽  
Covalently Linked ◽  
Von Willebrand ◽  
Willebrand Factor

While general mechanisms by which Plasmodium ookinetes invade the mosquito midgut have been studied, details remain to be understood regarding the interface of the ookinete, specifically its barriers to invasion, such as the proteolytic milieu, the chitin-containing, protein cross-linked peritrophic matrix, and the midgut epithelium. Here we review knowledge of Plasmodium chitinases and the mechanisms by which they mediate the ookinete crossing the peritrophic matrix. The integration of new genomic insights into previous findings advances our understanding of Plasmodium evolution. Recently obtained Plasmodium spp. genomic data enable identification of the conserved residues in the experimentally demonstrated hetero-multimeric, high molecular weight complex comprised of a short chitinase covalently linked to binding partners, von Willebrand factor A domain-related protein (WARP) and secreted ookinete adhesive protein (SOAP). Artificial intelligence-based high-resolution structural modeling using the DeepMind AlphaFold algorithm yielded highly informative 3D structures and insights into how short chitinases, WARP, and SOAP may interact at the atomic level to form the ookinete-secreted peritrophic matrix invasion complex. Elucidating the significance of the divergence of ookinete-secreted micronemal proteins among Plasmodium species could lead to a better understanding of ookinete invasion machinery and the co-evolution of Plasmodium -mosquito interactions.

scholarly journals Ligands and Receptors Involved in the Sperm-Zona Pellucida Interactions in Mammals

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 133
Author(s):  
Lucie Tumova ◽  
Michal Zigo ◽  
Peter Sutovsky ◽  
Marketa Sedmikova ◽  
Pavla Postlerova
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Present Article ◽  
Zona Pellucida ◽  
Protein Complexes ◽  
Model Organisms ◽  
Carbohydrate Composition ◽  
Comprehensive Understanding ◽  
Sperm Surface ◽  
Complex Process

Sperm-zona pellucida (ZP) interaction, involving the binding of sperm surface ligands to complementary carbohydrates of ZP, is the first direct gamete contact event crucial for subsequent gamete fusion and successful fertilization in mammals. It is a complex process mediated by the coordinated engagement of multiple ZP receptors forming high-molecular-weight (HMW) protein complexes at the acrosomal region of the sperm surface. The present article aims to review the current understanding of sperm-ZP binding in the four most studied mammalian models, i.e., murine, porcine, bovine, and human, and summarizes the candidate ZP receptors with established ZP affinity, including their origins and the mechanisms of ZP binding. Further, it compares and contrasts the ZP structure and carbohydrate composition in the aforementioned model organisms. The comprehensive understanding of sperm-ZP interaction mechanisms is critical for the diagnosis of infertility and thus becomes an integral part of assisted reproductive therapies/technologies.

P4-129 Genetic study of APH-1 and PEN-2, components of presenilin high molecular weight complex

2004 ◽  
Vol 25 ◽  
pp. S512
Author(s):  
Toshitaka Kawarai ◽  
Antonio Orlacchio ◽  
Ekaterina Rogaeva ◽  
Susan Ling ◽  
Hiroshi Hasegawa ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Genetic Study ◽  
High Molecular Weight Complex
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