Transgenic pig carrying green fluorescent proteasomes

Abstract

Among its many functions, the ubiquitin-proteasome system regulates substrate-specific proteolysis during the cell cycle, apoptosis, and fertilization and in pathologies such as Alzheimer's disease, cancer, and liver cirrhosis. Proteasomes are present in human and boar spermatozoa, but little is known about the interactions of proteasomal subunits with other sperm proteins or structures. We have created a transgenic boar with green fluorescent protein (GFP) tagged 20S proteasomal core subunit α-type 1 (PSMA1-GFP), hypothesizing that the PSMA1-GFP fusion protein will be incorporated into functional sperm proteasomes. Using direct epifluorescence imaging and indirect immunofluorescence detection, we have confirmed the presence of PSMA1-GFP in the sperm acrosome. Western blotting revealed a protein band corresponding to the predicted mass of PSMA1-GFP fusion protein (57 kDa) in transgenic spermatozoa. Transgenic boar fertility was confirmed by in vitro fertilization, resulting in transgenic blastocysts, and by mating, resulting in healthy transgenic offspring. Immunoprecipitation and proteomic analysis revealed that PSMA1-GFP copurifies with several acrosomal membrane-associated proteins (e.g., lactadherin/milk fat globule E8 and spermadhesin alanine-tryptophan-asparagine). The interaction of MFGE8 with PSMA1-GFP was confirmed through cross-immunoprecipitation. The identified proteasome-interacting proteins may regulate sperm proteasomal activity during fertilization or may be the substrates of proteasomal proteolysis during fertilization. Proteomic analysis also confirmed the interaction/coimmunoprecipitation of PSMA1-GFP with 13/14 proteasomal core subunits. These results demonstrate that the PSMA1-GFP was incorporated in the assembled sperm proteasomes. This mammal carrying green fluorescent proteasomes will be useful for studies of fertilization and wherever the ubiquitin-proteasome system plays a role in cellular function or pathology.

Identification of bovine sperm acrosomal proteins that interact with a 32-kDa acrosomal matrix protein

“…They proposed that the C-terminal peptide of the C2-like domain lactadherin interacts with the sperm membrane during capacitation and the C2-like domain is homologous to the phosphatidylserine-binding protein region of factor VIII [57] and is highly conserved within the lactadherin family [58]. Utilizing proteomic and co-immunoprecipitation analyses, Miles et al [59] presented that green fluorescent protein (GFP) tagged 20S proteasomal core subunit α-type1 (PSMA1-GFP) interacts with several acrosomal membrane-associated proteins of pig sperm; one of the proteins is lactadherin. They proposed that lactadherin anchors proteasomes to the acrosomal structure and exhibits in proteasome-assisted acrosomal remodeling during sperm capacitation and acrosomal exocytosis.…”

Sperm Proteasome as a Putative Egg Coat Lysin in Mammals

“…Depletion of sperm surface ATP by Solanum tuberosum apyrase inhibits sea urchin fertilization (Yokota and Sawada 2007 ) and porcine in vitro fertilization (IVF) (Yi et al 2009 ). Enzymatic activity of boar sperm proteasomes was confi rmed in whole, motile spermatozoa, sperm acrosomal fractions, and affi nity purifi ed proteasomes (Yi et al 2009 ;Miles et al 2013 ). These purifi ed proteasomes were also tested for functionality via casein degradation (Manandhar and Sutovsky, unpublished data).…”

“…A novel transgenic boar model with green fl uorescent protein (GFP) fused to the C-terminus of 20S proteasomal core subunit alpha-type 6 (PSMA1) has been developed through a joint effort between the Sutovsky, Prather, and Wells laboratories at the University of Missouri-Columbia (Miles et al 2013 ). Functional GFP-tagged proteasomes have been shown to be incorporated in not only fertilization-competent spermatozoa but in other tissues and cell types (Miles et al 2013 ).…”

“…Functional GFP-tagged proteasomes have been shown to be incorporated in not only fertilization-competent spermatozoa but in other tissues and cell types (Miles et al 2013 ). Using crossimmunoprecipitation experiments, the authors identifi ed various proteins interacting with the GFP-PSMA1 subunit such as lactadherin/MFGE8, spermadhesins, and disintegrins/ADAM metalloproteinases; these proteins may regulate sperm proteasomal activity or may be the substrates of proteasomal proteolysis during fertilization (Miles et al 2013 ). They have also proposed a method of isolating enzymatically active GFP proteasomes through GFP affi nity purifi cation (Miles et al 2013 ).…”

“…Using crossimmunoprecipitation experiments, the authors identifi ed various proteins interacting with the GFP-PSMA1 subunit such as lactadherin/MFGE8, spermadhesins, and disintegrins/ADAM metalloproteinases; these proteins may regulate sperm proteasomal activity or may be the substrates of proteasomal proteolysis during fertilization (Miles et al 2013 ). They have also proposed a method of isolating enzymatically active GFP proteasomes through GFP affi nity purifi cation (Miles et al 2013 ). This novel model will be useful for studies of fertilization and wherever UPS plays a role in cellular function or pathology.…”