leupeptins and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

We previously reported that sperm proteasome is responsible for degradation of the ubiquitinated vitelline-coat during fertilization in the ascidian Halocynthia roretzi. Here, we report the roles in fertilization and localization in the sperm cell surface of H. roretzi sperm proteasome. An anti-proteasome antibody, as well as the proteasome inhibitors MG115 and MG132, inhibited the fertilization, indicating that the sperm proteasome functions extracellularly in ascidian fertilization. In order to further assess this issue, the sperm surface proteasome activity was labeled with a cell-impermeable labeling reagent, NHS-LC-biotin, extracted with 0.1% CHAPS, and was subjected to a pull-down assay with avidin-agarose beads. It was found that a substantial amount of sperm proteasome is exposed to the cell surface. Partition analysis with Triton X-114 also revealed that a considerable amount of the sperm proteasome activity is partitioned into a lipid layer. Localization of the proteasome activity was investigated by fluorescence microscopy with succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide as a substrate. The sperm proteasome activity was specifically detected in the sperm head region, and it was markedly activated upon sperm activation. The membrane-associated proteasome was purified from the membrane fraction of H. roretzi sperm by affinity chromatography using an anti-20S proteasome antibody-immobilized Sepharose column. SDS-PAGE of the purified preparation showed a similar pattern of subunit composition to that of the 26S proteasome of mammalian origin. Taken together, these results indicate that H. roretzi sperm has the membrane-associated proteasome on its head, which is activated upon sperm activation, and that sperm proteasome plays an essential role in H. roretzi fertilization.

Topics: Animals; Cell Membrane; Cholic Acids; Cysteine Proteinase Inhibitors; Detergents; Enzyme Activation; Female; Fertilization; Leupeptins; Male; Peptide Hydrolases; Proteasome Endopeptidase Complex; Spermatozoa; Urochordata

The MHC class II alpha beta heterodimer associates with invariant (I) chain in the endoplasmic reticulum and remains associated until the complex reaches a post-Golgi compartment. During early stages of transport, I chain blocks peptide binding to alpha beta dimers. I chain is proteolytically cleaved in a post-Golgi compartment releasing alpha beta dimers that can bind antigenic peptides and transport them to the cell surface. Human B lymphoblastoid cell lines grown in leupeptin, a sulfhydryl protease inhibitor, accumulate a partial proteolytic product of the I chain called leupeptin-induced protein (LIP). LIP remains associated with alpha beta dimers. We find, using chemical cross-linking, sucrose gradient sedimentation, and size exclusion chromatography, that the alpha beta LIP complex retains the nine-subunit structure described for alpha beta I complexes. Unlike the alpha beta I complex, in certain detergents the alpha beta LIP nonamer is unstable and dissociates into trimers containing one alpha, beta, and LIP molecule. This finding emphasizes the reported stoichiometry of the alpha beta I complex as a nine-subunit structure comprised of three alpha beta I trimers. Also, these data indicate that the region(s) of I chain necessary for retaining the nonameric structure lie within the LIP fragment, but that domains to the C-terminus of the LIP cleavage site act to further stabilize the nine chain structure. In addition, alpha beta I complexes containing forms of human I chain encoding the p35/p43 N-terminal cytoplasmic extension responsible for endoplasmic reticulum retention can transport to post-Golgi proteolytic compartments where LIP is formed.

Topics: Antigens, Differentiation, B-Lymphocyte; Biological Transport; Cell Line; Centrifugation, Density Gradient; Cholic Acids; Chromatography, Gel; Histocompatibility Antigens Class II; Humans; Leupeptins; Molecular Weight; Peptide Fragments