Fertilin ␣ (also known as ADAM1) is a member of the ADAM (A disintegrin and A metalloprotease domain) family of proteins. In this study, we examine the mechanism of mouse fertilin ␣'s in adhesion of sperm to the egg plasma membrane during fertilization. We find that recombinant forms of fertilin ␣ corresponding to either the disintegrin-like domain or the cysteine-rich domain and the EGF-like repeat can perturb sperm-egg binding, suggesting that both of these domains can participate in fertilin ␣-mediated adhesion events. In further examination of the fertilin ␣ disintegrin-like domain, we find that a subdomain of disintegrin-like domain with the sequence DLEECDCG outside the putative disintegrin loop but with homology to the fertilin ␤ disintegrin loop can inhibit the binding of both sperm and recombinant fertilin ␣ to eggs, suggesting that this is an adhesionmediating motif of the fertilin ␣ disintegrin-like domain. This sequence also inhibits the binding of recombinant fertilin ␤ to eggs and thus is the first peptide sequence found to block two different sperm ligands. Finally, a monoclonal antibody to the tetraspanin protein CD9, KMC.8, inhibited the binding of recombinant fertilin ␣ to eggs in one type of binding assay, suggesting that, under certain conditions, fertilin ␣ may interact with a KMC.8-sensitive binding site on the egg plasma membrane. ADAM1 (for A disintegrin and A metalloprotease) proteins comprise a recently identified and rapidly growing molecular family of membrane proteins. To date, ϳ30 ADAMs have been identified in a variety of animal species, including several mammals, Xenopus laevis, Drosophila melanogaster, and Caenorhabditis elegans (1, 2). The conserved domain structure of ADAMs (see Fig. 1A) and functional analyses of these proteins indicate that members of this family have functions as proteases and/or as cell adhesion molecules. In the ADAMs that have been described to function as cell adhesion molecules, the adhesive activity generally appears to be attributable to the disintegrin-like domain, a domain with homology to integrin ligand-like snake venom polypeptides. These venom polypeptides, known as disintegrins, contain an RGD tripeptide, presented at the end of an extended loop structure called the "disintegrin loop" (3). Although ADAMs share significant sequence homology in their disintegrin-like domains to snake venom disintegrins, they do not have RGD sequences in their putative disintegrin loops (with the exception of human ADAM15). Various studies have shown that the adhesion-mediating sequence of several ADAMs appears to be a short sequence within the putative disintegrin loop, such as the ECDcontaining sequences in fertilin ␤ (4, 5), ADAM9 (6), and ADAM23 (7), or the QCD-containing sequence in cyritestin (8).Among the cell adhesion events that are mediated by ADAMs are the interactions between mammalian gamete plasma membranes during fertilization. On mouse sperm, there are at least three ADAMs that appear to participate in sperm-egg binding: fertilin ␣ (ADAM1), fertili...

Antisense oligo-2'-O-methylribonucleotides and their methylphosphonate derivatives show high binding affinities for their complementary targets under essentially physiological conditions. Additionally, the methylphosphonate linkage is resistant to nuclease hydrolysis. Here we show that a single methylphosphonate internucleotide linkage at the 3'-end of an oligo-2'-O-methylribonucleotide is sufficient to prevent degradation by the 3'-exonuclease activity found in mammalian serum. Complexes formed between a cationic lipid, Oligofectamine, and 5'-[(32)P]-labeled methylphosphonate modified oligo-2'-O-methylribonucleotides are taken up by mouse L(929) fibroblasts in culture. The extent of uptake appears to be dependent upon the sequence of the oligonucleotide. Examination of lysates of oligonucleotide treated cells by polyacrylamide gel electrophoresis showed that no degradation of the oligonucleotide occurred, even after incubation for 24 h. A fluorescein-derivatized oligomer was shown to localize mainly in the cell nucleus as monitored by fluorescence microscopy. Covalent conjugates of fluorescein-derivatized 3'-methylphosphonate modified oligo-2'-O-methylribonucleotides with Tat peptide, a cell permeating peptide, were also prepared. The Tat peptide was coupled to the 5'-end of the oligonucleotide using either disulfide coupling chemistry or conjugation of a keto derivative of the Tat peptide via a 4-(2-aminooxyethoxy-2-(ethylureido)quinoline group at the 5'-end of the oligonucleotide. Although formation of the Tat peptide conjugates was confirmed by mass spectrometry, the propensity of these oligonucleotides to form aggregates and their apparent high affinity for plastic and glass made the conjugates unsuitable for studies of uptake by cells in culture.

Chimeric oligo-2'-O-methylribonucleotides containing centrally located patches of contiguous 2'-deoxyribonucleotides and terminating in a nuclease resistant 3'-methylphosphonate internucleotide linkage were prepared. The oligonucleotides were targeted to the 3'-side of HIV Rev response element (RRE) stem-loop IIB RNA, which is adjacent to the high affinity Rev protein binding site and is critical to virus function. Thermal denaturation experiments showed that chimeric oligonucleotides form very stable duplexes with a complementary single-stranded RNA, and gel electrophoretic mobility shift assays (EMSA) showed that they bind with high affinity and specificity to RRE stem-loop II RNA (K(D) approximately 200 nM). The chimeric oligonucleotides promote RNase H-mediated hydrolysis of RRE stem-loop II RNA and have half-lives exceeding 24h when incubated in cell culture medium containing 10% fetal calf serum. One of the chimeric oligonucleotides inhibited RRE mediated expression of chloramphenicol acetyl transferase (CAT) approximately 60% at a concentration of 300 nM in HEK 293T cells co-transfected with p-RRE/CAT and p-Rev mammalian expression vectors.