The Chesapeake Bay — A Classroom for Gifted Students

105

A hallmark of gamete interactions at fertilization is relative or absolute species specificity. A pig sperm protein that binds to the extracellular matrix of the egg in a species-specific manner was recently identified and named zonadhesin (Hardy, Prior to fertilization, the sperm cell interacts with multiple cells including fellow sperm cells, Sertoli cells, epithelial cells within the male reproductive tract or female reproductive tract, cells associated with the egg, and the egg itself. In some cases, the interactions are attractive in nature and in others repulsive. In an attempt to identify sperm proteins that bind to the egg extracellular matrix, Hardy and Garbers (1) solubilized sperm membranes from the pig and determined which proteins bound to the pig zona pellucida. Subsequently, the cDNA encoding a protein that bound to the zona pellucida in a relatively species-specific manner was cloned and named zonadhesin (2). Pig zonadhesin is a transmembrane protein with a very short intracellular region. The putative extracellular region contains a mucin-like domain and five tandem repeats homologous to the D-domains of prepro-von Willebrand factor (ppvWF) 1 (3, 4). The cloning of the pig sperm zonadhesin cDNA raised the questions of whether other species would contain homologs of zonadhesin and, if so, whether these would be variable within the above domain structure. Here, the mouse homolog of zonadhesin is cloned and shown to contain additional repetitive sequences within the D-domain region. Furthermore, three tandem repeats of a MAM domain were identified at the N terminus. The MAM domain is a module composed of about 160 amino acids including four conserved Cys; it is found in a number of functionally diverse transmembrane proteins such as the meprins and receptor protein-tyrosine phosphatases (5). Thus, zonadhesin displays a large species variation between pig and mouse and also contains multiple domains previously suggested as involved in cell adhesion processes.D MATERIALS AND METHODSRNA Isolation-Total RNA was isolated from 40 mouse testes or similar amounts of other mouse tissues (including brain, heart, kidney, liver, lung, small intestine, and spleen) in guanidinium thiocyanate and N-lauroyl sarcosine, followed by extraction with acidic phenol/CHCl 3 and precipitation with isopropyl alcohol (6). Poly(A) ϩ RNA was purified from total RNA by oligo(dT)-cellulose chromatography (7).Northern Blots-30 g of total RNA from various mouse tissues was loaded on each lane of a formaldehyde-agarose gel (8) and blotted overnight using 10 ϫ SSC as the transfer buffer. The blot was then fixed by UV light and hybridized with a random primer-labeled (Amersham Life Science, Inc.), 1.4-kb probe from the 3Ј-end of the mouse zonadhesin cDNA as described previously (1). After hybridization, the blot was washed with 0.2 ϫ SSC and 0.2% SDS at 65°C.Screening of cDNA Libraries-The first mouse cDNA clone (#18) was obtained by a low stringency screening of a mouse testis cDNA library (Stratagene) using a cDNA fragment correspo...

Section: Resultsmentioning

confidence: 99%

Species Diversity in the Structure of Zonadhesin, a Sperm-specific Membrane Protein Containing Multiple Cell Adhesion Molecule-like Domains

Gao¹,

Garbers²

1998

105

“…Interestingly, no other protein domain data base and search tool predicted this Type II EGF-like signature. Although the precise roles of the TSR and potential EGF domains are as yet unclear, these domains are located in the extracellular region of membrane-bound proteins or in proteins known to be secreted (29). Although TSR (13)(14)(15) or EGF domains (30 -32) are individually present in a number of Plasmodium proteins, no Plasmodium antigen has been reported in which these two domains are predicted to be present together.…”

Section: Reactivity Of Sera From Malaria Endemic Regions Withmentioning

confidence: 99%

PfSPATR, a Plasmodium falciparum Protein Containing an Altered Thrombospondin Type I Repeat Domain Is Expressed at Several Stages of the Parasite Life Cycle and Is the Target of Inhibitory Antibodies

Chattopadhyay¹,

Rathore

Fujioka

et al. 2003

The annotated sequence of chromosome 2 of Plasmodium falciparum was examined for genes encoding proteins that may be of interest for vaccine development. We describe here the characterization of a protein with an altered thrombospondin Type I repeat domain (PfSPATR) that is expressed in the sporozoite, asexual, and sexual erythrocytic stages of the parasite life cycle. Immunoelectron microscopy indicated that this protein was expressed on the surface of the sporozoites and around the rhoptries in the asexual erythrocytic stage. An Escherichia coli-produced recombinant form of the protein bound to HepG2 cells in a dose-dependent manner and antibodies raised against this protein blocked the invasion of sporozoites into a transformed hepatoma cell line. Sera from Ghanaian adults and from a volunteer who had been immunized with radiation-attenuated P. falciparum sporozoites specifically recognized the expression of this protein on transfected COS-7 cells. These data support the evaluation of this protein as a vaccine candidate.

“…Consensus sequence domains contain the amino acids Asp, Asp/Asn, Asp/ Asn, and Tyr/Phe at defined positions. The alignment of these latter four residues are thought to signal post-translational hydroxylation of the third site in the consensus by BAH (4). The consensus sequence for aspartyl ␤-hydroxylation has been identified in a diverse group of proteins including clotting factors (5), Notch receptors and ligands (6 -8), in structural proteins of the extracellular matrix (9), and in ligands of the tyro-3/Axl family of receptor tyrosine kinases (10).…”

mentioning

confidence: 99%

Aspartyl β-Hydroxylase (Asph) and an Evolutionarily Conserved Isoform of Asph Missing the Catalytic Domain Share Exons with Junctin

Dinchuk¹,

Henderson²,

Burn³

et al. 2000

146

The mouse aspartyl ␤-hydroxylase gene (Asph, BAH) has been cloned and characterized. The mouse BAH gene spans 200 kilobase pairs of genomic DNA and contains 24 exons. Of three major BAH-related transcripts, the two largest (6,629 and 4,419 base pairs) encode fulllength protein and differ only in the use of alternative polyadenylation signals. The smallest BAH-related transcript (2,789 base pairs) uses an alternative 3 terminal exon, resulting in a protein lacking a catalytic domain. Evolutionary conservation of this noncatalytic isoform of BAH (humbug) is demonstrated in mouse, man, and Drosophila. Monoclonal antibody reagents were generated, epitope-mapped, and used to definitively correlate RNA bands on Northern blots with protein species on Western blots. The gene for mouse junctin, a calsequestrin-binding protein, was cloned and characterized and shown to be encoded from the same locus. When expressed in heart tissue, BAH/humbug preferably use the first exon and often the fourth exon of junctin while preserving the reading frame. Thus, three individual genes share common exons and open reading frames and use separate promoters to achieve differential expression, splicing, and function in a variety of tissues. This unusual form of exon sharing suggests that the functions of junctin, BAH, and humbug may be linked.