Thermal Spray Copper Alloy Coatings as Potent Biocidal and Virucidal Surfaces.

Microbial and viral pathogen contamination of touch surfaces contributes to the rapid transmission of diseases. It has been known for decades that microbes and viruses are rapidly inactivated when exposed to copper and its alloys. Consequently, the use of thermal spray technologies to coat surfaces in healthcare and public settings has been receiving a considerable amount of interest during recent viral pandemics and particularly now with COVID-19. This review is focused on recent successes using thermal spray technology to uniformly coat metal and organic surfaces, providing a rapid and economical means of inhibiting fomite transmission of pathogens on diverse surfaces with complex topographies. Emphasis is placed on the influence of lamella structure, porosity, and roughness of the coatings as it pertains to biocidal activity and the implications of using this knowledge to optimize the ability of copper coatings to irreversibly inactivate viral pathogens, regardless of their genomic mutation rates. Results of the long-term performance of the copper alloy coatings in real hospital settings in Canada and Peru are also presented.

Evaluation of biocidal efficacy of copper alloy coatings in comparison with solid metal surfaces: generation of organic copper phosphate nanoflowers.

AIMS: To analyse the biocidal efficacy of thermal sprayed copper surfaces. METHODS AND RESULTS: Copper alloy sheet metals containing >60% copper have been shown to exhibit potent biocidal activity. Surface biocidal activity was assessed by epifluorescence microscopy. After 2-h exposure at 20 °C in phosphate-buffered saline (PBS), contact killing of Gram-negative Escherichia coli and Gram-positive Staphylococcus epidermidis by brass sheet metal and phosphor bronze was 3-4-times higher than that by stainless steel. SEM observations revealed that the surface membranes of both bacterial strains were slightly more irregular when exposed to brass sheet metal than stainless steel. However, when exposed to phosphor bronze coating, E. coli were 3-4 times larger with irregular membrane morphology. In addition, the majority of the cells were associated with spherical carbon-copper-phosphate crystalline nanostructures characteristic of nanoflowers. The membranes of many of the S. epidermidis exhibited blebbing, and a small subset was also associated with nanoflowers. CONCLUSIONS: Our data indicate that increasing the surface roughness of copper alloys had a pronounced impact on the membrane integrity of Gram-positive and, to a lesser degree, Gram-negative bacteria. In the presence of PBS, carbon-copper-phosphate-containing nanoflowers were formed, likely nucleated by components derived from killed bacteria. The intimate association of the bacteria with the nanoflowers and phosphor bronze coating likely contributed to their nonreversible adhesion. SIGNIFICANCE AND IMPACT OF THE STUDY: Thermal spraying of copper alloys provides a strategy for the rapid coating of three-dimensional organic and inorganic surfaces with biocidal copper alloys. Our study demonstrates that the macroscale surface roughness generated by the thermal spray process enhances the biocidal activity of copper alloys compared with the nanoscale surface roughness of copper sheet metals. Moreover, the coating surface topography provides conditions for the rapid formation of organic copper phosphate nanocrystals/nanoflowers.

Phagocytosis of collagen by fibroblasts and invasive cancer cells is mediated by MT1-MMP.

Degradation of collagen is required for the physiological remodelling of connective tissues during growth and development, as well as in wound healing, inflammatory diseases, and cancer cell invasion. In remodelling adult tissues, degradation of collagen occurs primarily through a phagocytic pathway. While various steps in this pathway have been characterized, the enzyme required to fragment collagen fibrils for phagocytosis has not been identified. Laser confocal microscopy, transmission electron microscopy and biochemical assays were used to show that degradation of collagen substrates by fibroblasts correlated with the expression of the membrane-bound metalloproteinase MT1-MMP (membrane-type 1 matrix metalloproteinase). The MT1-MMP was localized to sites of collagen cleavage on the cell surface and also within the cells. In contrast with MT1-MMP, the gelatinase MMP-2 was not required for collagen phagocytosis. Similar analyses of several ovarian cancer, breast cancer and fibrosarcoma cells indicated that highly metastatic cells also degrade collagen through a phagocytic pathway that is mediated by MT1-MMP. Collectively, these studies demonstrate a pivotal role for catalytically active MT1-MMP in preparing collagen fibrils for phagocytic degradation by normal and transformed cells.

MT1-MMP is the critical determinant of matrix degradation and invasion by ovarian cancer cells.

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane metalloprotease that plays an important role in the invasion of many solid tumour types, promotes pericellular matrix degradation and may also stimulate tumour cell motility. As both these processes are key contributors to intraperitoneal ovarian tumour metastasis, we examined six ovarian cancer cell lines to determine whether MT1 is a critical mediator of invasive behaviour for this tumour type. Our results indicated that only those cell lines that expressed MT1 were capable of penetrating a type I collagen barrier, with the capacity for both matrix degradation and invasion reflecting endogenous MT1 expression level. Ectopic MT1 expression endowed an invasive phenotype upon cell lines lacking MT1 that were previously non-invasive, indicating the crucial role of this protease. Conversely, invasion was abolished by tissue inhibitor of metalloproteinase-2 (TIMP-2), a potent inhibitor of MT1, yet was minimally affected when other (secreted) MMPs were inhibited using TIMP-1 and the gelatinase inhibitor SB-3CT. Whereas collagen I degradation was strikingly accelerated by ectopic MT1 expression, cell motility remained unchanged. We conclude that MT1 is necessary for collagen I invasion by ovarian cancer cells, and that its requisite activity is the promotion of matrix degradation, with no impact on cell motility.

Is SPARC an evolutionarily conserved collagen chaperone?

The construction of collagen fiber scaffolds, which provide the structural integrity of the extracellular matrix of connective tissues and basement membranes, is initiated by a complex mechanism of protein-folding, whereby pro-collagen alpha-chains are assembled into triple-helical procollagen molecules. This unique assembly of the procollagen molecules is guided by several endoplasmic reticulum resident molecular chaperones, including HSP47, which dissociates from procollagen molecules prior to their transport from the endoplasmic reticulum into the cis-Golgi network. SPARC, an evolutionarily conserved collagen-binding glycoprotein, which is frequently co-expressed with collagen in rapidly remodeling tissues, binds to the triple-helical region of procollagen molecules. Analysis of data from genome projects indicates that specific amino acids and sequences in SPARC that are critical for collagen binding are evolutionarily conserved in organisms ranging from nematodes to mammals. Studies of invertebrates, which do not encode HSP47, indicate that SPARC expression is required for the deposition of collagen IV in basal lamina during embryonic development. In mammals, defects in collagen deposition have been observed in normal and wound-healing tissues in the absence of SPARC expression. Based on these and other observations, we propose that intracellular SPARC acts as a collagen molecular chaperone in the endoplasmic reticulum, and that in higher organisms, SPARC acts in concert with HSP47 to ensure that only correctly folded procollagen molecules exit the endoplasmic reticulum. In contrast to HSP47, SPARC is transported from the endoplasmic reticulum through the Golgi network and into secretory vesicles for exocytosis at the plasma membrane. Hence, SPARC may also play a role in regulating post-endoplasmic reticulum events that promote collagen fibrillogenesis.

Association of SPARC (osteonectin, BM-40) with extracellular and intracellular components of the ciliated surface ectoderm of Xenopus embryos.

SPARC (Secreted Protein, Acidic, Rich in Cysteine) was detected by immunohistochemistry in the sensorial layer of the bilayered embryonic epidermis of Xenopus laevis during neurulation, when a subset of the sensorial cells are selected to differentiate into ciliated cell precursors. After the ciliated cells had intercalated into the outer layer and had undergone ciliogenesis, intense SPARC immunostaining was associated with the cilia and remained associated with the cilia throughout their persistence on the epidermis. Circumferential SPARC immunostaining was also detected at the interface between surface epithelial cells. Animal cap explants indicated that the embryonic activation of SPARC expression in the dorsal ectoderm does not require signaling from factors secreted by the underlying mesoderm. Immunoelectron microscopy revealed that SPARC is intimately associated with the 9 + 2 microtubule arrays of cilia. Our data indicate that SPARC plays a role in the development and function of the surface ciliated epidermis of Xenopus embryos. We propose that the counter-adhesive activity of SPARC facilitates the intercalation of ciliary cell precursors to the surface epithelial layer, where its Ca(2+)-binding abilities promote cell-cell adhesion. Based on its association with ciliary microtubule arrays, we also propose that intracellular SPARC may play a role in regulating ciliary beat frequency and polarity.

Activation of SPARC expression in reactive stroma associated with human epithelial ovarian cancer.

OBJECTIVE: SPARC (secreted protein, acidic, rich in cysteine) is a calcium-binding counteradhesive glycoprotein that has the potential to play an important role in promoting tumor progression and invasiveness. SPARC has been reported to be markedly down-regulated in ovarian carcinomas relative to the normal surface epithelium and has been suggested to act as a tumor suppressor in ovarian cancer. To more precisely define potential changes in SPARC expression associated with malignant transformation of the ovary, we compared the distribution of SPARC mRNA and protein expression in patient specimens of malignant and nonmalignant ovaries. METHOD: SPARC mRNA and protein expression was examined in 24 human invasive ovarian cancers, 5 tumors of low malignant potential (LMP), and 8 nonmalignant ovaries by in situ hybridization and immunohistochemistry. RESULTS: In nonmalignant ovaries, SPARC mRNA expression was restricted to thecal and granulosa cells of vessiculated follicles. Cytoplasmic SPARC immunoreactivity was observed in these compartments, whereas variable SPARC immunostaining was observed in normal surface epithelial cells. In contrast, high-level expression of SPARC mRNA and protein was detected in stroma of ovaries containing malignant tumor cells, particularly at the tumor-stromal interface of the invading tumors. Lower levels and a more diffuse pattern of SPARC mRNA expression were associated with LMP specimens. SPARC mRNA was not expressed by ovarian adenocarcinoma or by surface epithelial cells. Consistent with the in situ hybridization data, SPARC immunoreactivity was found throughout the reactive stroma of specimens containing ovarian carcinoma. However, despite the lack of detectable SPARC mRNA, SPARC immunoreactivity was consistently observed within the cytoplasm of cancer cells. CONCLUSION: The pattern of SPARC expression shown in this study indicates that SPARC is up-regulated in reactive stroma associated with invasive ovarian cancer. Moreover, these results raise the possibility that SPARC secreted from the stroma is internalized by ovarian cancer cells and may exert important intracellular effects upon these cells.

A calcium-binding motif in SPARC/osteonectin inhibits chordomesoderm cell migration during Xenopus laevis gastrulation: evidence of counter-adhesive activity in vivo.

Secreted protein, acidic, rich in cysteine (SPARC) is a Ca2+-binding, counter-adhesive, extracellular glycoprotein associated with major morphogenic events and tissue remodeling in vertebrates. In Xenopus laevis embryos, SPARC is expressed first by dorsal mesoderm cells at the end of gastrulation and undergoes complex, rapid changes in its pattern of expression during early organogenesis. Another study has reported that precocious expression of SPARC by injection of native protein into the blastocoele cavity of pregastrula embryos leads to a concentration-dependent reduction in anterior development. Thus, normal development requires that the timing, spatial distribution, and/or levels of SPARC be regulated precisely. In a previous study, we demonstrated that injection of a synthetic peptide corresponding to the C-terminal, Ca2+-binding, EF-hand domain of SPARC (peptide 4.2) mimicked the effects of native SPARC. In the present investigation, peptide 4.2 was used to examine the cellular and molecular bases of the phenotypes generated by the aberrant presence of SPARC. Exposure of late blastula embryos to LiCl also generated a concentration-dependent reduction in anterior development; therefore, injections of LiCl were carried out in parallel to highlight the unique effects of peptide 4.2 on early development. At concentrations that caused a similar loss in anterior development (60-100 ng peptide 4.2 or 0.25-0.4 microg LiCl), LiCl had a greater inhibitory effect on the initial rate of chordomesoderm cell involution, in comparison with peptide 4.2. However, as gastrulation progressed, peptide 4.2 had a greater inhibitory effect on prospective head mesoderm migration than that seen in the presence of LiCl. Moreover, peptide 4.2 and LiCl had distinct influences on the expression pattern of dorso-anterior markers at the neural and tail-bud stages of development. Scanning electron microscopy showed that peptide 4.2 inhibited spreading of migrating cells at the leading edge of the involuting chordomesoderm. While still in close proximity to the blastocoele roof, many of the cells appeared rounded and lacked lamellipodia and filopodia extended in the direction of migration. In contrast, LiCl had no effect on the spreading or shape of involuting cells. These data are the first evidence of a counter-adhesive activity for peptide 4.2 in vivo, an activity demonstrated for both native SPARC and peptide 4.2 in vitro.

Expression of SC1 is associated with the migration of myotomes along the dermomyotome during somitogenesis in early mouse embryos.

SC1 is a secreted glycoprotein with a high amino acid sequence similarity to SPARC (Secreted Protein, Acidic, Rich in Cysteine). SC1 transcripts were first detected in mouse embryos after day 8.5 post coitus (p.c.) in somites at the medial lip of the dermomyotome. Expression of SC1 transcripts by the progenitor cells continued as they began involuting under the dermomyotome and during their migration along the lateral wall of the dermomyotome. After myotome migration was completed, SC1 mRNA expression was downregulated in the trunk region. The data indicate that SC1 expression is restricted to the initial stages of epaxial myotome differentiation and migration, undergoing rapid downregulation prior to myotome emigration from the somitic environment.

Regulation of SPARC expression during early Xenopus development: evolutionary divergence and conservation of DNA regulatory elements between amphibians and mammals.

SPARC (Secreted Protein, Acidic, Rich in Cysteine/osteonectin/BM-40) is a highly conserved metal-binding extracellular matrix (ECM) glycoprotein which is first expressed by Xenopus embryos during late gastrulation/early neurulation (stage 12/13), by presumptive notochord and somitic cells. When animal cap explants of stage 9 embryos were cultured in vitro, SPARC expression was not detected until sibling embryos reached late neurula stage (stage 19). Addition of activin, a potent dorsal mesoderm inducer, to animal caps resulted in SPARC being expressed by the time sibling embryos reached stage 16. While basic fibroblast growth factor (bFGF), a ventral mesoderm inducer, had modest effects on SPARC mRNA expression, the combination of both activin and bFGF was synergistic. The appearance, however, of SPARC transcripts 11 h after the addition of activin and bFGF, indicates that unknown intermediates were likely to be involved in activating SPARC expression. In order to identify the potential intermediate regulatory factors which may activate and control SPARC expression, we examined the genomic organization of the 5' end of the Xenopus SPARC gene. No significant homology to the equivalent region that is highly conserved in the mouse, bovine and human SPARC genes was observed. Thus, while mammalian SPARC promoters lack TATA or CAAT boxes, the Xenopus gene contains a consensus TATA box. Moreover, promoter-proximal GGA-box repeats necessary for high level expression of mammalian SPARC are absent in Xenopus. When reporter constructs containing the 5' flanking region of the Xenopus gene were microinjected into two-cell embryos, 868 bp of 5' flanking DNA was sufficient to mimic the temporal and tissue-specific pattern of SPARC expression observed in whole embryos. While a bovine SPARC promoter reporter construct containing 740 bp of the 5' flanking DNA was expressed at a significant level in Xenopus embryos, significant differences in the cell-type expression of the reporter genes were obtained between the bovine and Xenopus constructs. The data indicate that zygotic activation of SPARC mRNA is mediated by regulatory factors acting downstream of major mesoderm induction events. The high DNA sequence conservation at the 5' end of mammalian SPARC genes is not conserved in Xenopus. These differences led to differences in their ability to direct tissue-specific gene expression in early Xenopus embryos.

Ectopic expression of SPARC in Xenopus embryos interferes with tissue morphogenesis: identification of a bioactive sequence in the C-terminal EF hand.

SPARC is a matricellular Ca(2+)-binding glycoprotein that exhibits both counteradhesive and antiproliferative effects on cultured cells. It is secreted by cells of various tissues as a consequence of morphogenesis, response to injury, and cyclic renewal and/or repair. In an earlier study with Xenopus embryos we had shown a highly specific and regulated pattern of SPARC expression. We now show that ectopic expression of SPARC before its normal embryonic activation produces severe anomalies, some of which are consistent with the functions of SPARC proposed from studies in vitro. Microinjection of SPARC RNA, protein, and peptides into Xenopus embryos before endogenous embryonic expression generated different but overlapping phenotypes. (a) Injection of SPARC RNA into one cell of a two-cell embryo resulted in a range of unilateral defects. (b) Precocious exposure of embryos to SPARC by microinjection of protein into the blastocoel cavity was associated with certain axial defects comparable to those obtained with SPARC RNA. (c) SPARC peptides containing follistatin-like and copper-binding sequences were without obvious effect, whereas SPARC peptide 4.2, corresponding to a disulfide-bonded, Ca(2+)-binding domain, was associated with a reduction in axial structures that led eventually to complete ventralization of the embryos. Histological analysis of ventralized embryos indicated that the morphogenetic events associated with gastrulation might have been inhibited. Microinjection of other Ca(2+)-binding glycoproteins, such as osteopontin and bone sialoprotein, resulted in phenotypes that were unique. We probed further the structural correlates of this region of SPARC in the context of tissue development. Co-injection of peptide 4.2 with Ca2+ or EGTA, and injection of peptide 4.2K (containing a mutated consensus Ca(2+)-binding sequence), demonstrated that the developmental defects associated with peptide 4.2 were independent of Ca2+. However, the disulfide bridge in this region of SPARC was found to be critical, as injection of peptide 4.2AA, a mutant lacking the cystine, generated no axial defects. We have therefore shown for the first time in vivo that the temporally inappropriate presence of SPARC is associated with perturbations in tissue morphogenesis. Moreover, we have identified at least one bioactive region of SPARC as the C-terminal disulfide-bonded, Ca(2+)-binding loop that was previously shown to be both counteradhesive and growth-inhibitory.

Spatiotemporal distribution of SPARC/osteonectin in developing and mature chicken retina.

Expression of SPARC (Secreted Protein, Acidic, Rich in Cysteine), a counteradhesive, calcium-binding extracellular matrix (ECM) glycoprotein, is associated with several morphogenetic events during early development. In this study, changes in the spatiotemporal distribution of SPARC transcripts and the protein during chicken retinal development were documented by in situ hybridization and indirect immunofluorescence microscopy. SPARC transcripts were first detected within the proliferating neural ectoderm at embryonic day 4. 5 (E4.5), followed short thereafter (E5) by appearance of SPARC. SPARC was enriched within the inner plexiform layer (IPL) by E10 and within the outer plexiform layer (OPL) by E14, several days after these layers became morphologically distinct. Significant levels of SPARC transcripts were first observed within the ganglion cell layer (GCL) at E17 prior to accumulation of SPARC within the nerve fiber layer, seen first at E20. SPARC protein was first detected within the developing retinal pigment epithelium (RPE) at E10 and increased significantly at RPE cells ceased to proliferate and continued differentiating. Of special note was the restriction of SPARC to the basal-half of the RPE cells. SPARC transcripts were similarly distributed in the adult retina, but at lower levels than in the period just prior to hatching. In the adult retina SPARC was retained in the nerve fiber layer and present in the inner nuclear layer (INL) and outer nuclear layer (ONL), but lost from the IPL and OPL. These changes in expression pattern with time indicate that SPARC is developmentally regulated and therefore may have important function(s) in both morphological development of the retina and functioning of the mature eye.

Transient expression of SPARC in the dorsal axis of early Xenopus embryos: correlation with calcium-dependent adhesion and electrical coupling.

Our comprehension of the molecular mechanisms underlying embryogenesis has been greatly enhanced by the identification and characterization of associated extracellular matrix macromolecules. Using Xenopus laevis as a model, we investigated the expression and distribution of SPARC (Secreted Protein, Acidic, Rich in Cysteine; also called osteonectin and BM-40) during early embryonic development. SPARC has been found to be enriched in tissues undergoing rapid morphological development, differentiation, and remodeling. In Xenopus, SPARC transcripts are first expressed by primordial cells which give rise to the first embryonic tissues, the notochord and somites. SPARC RNA levels remained high throughout the rapid morphological development and differentiation phase of these tissues, and then rapidly decreased. Of particular interest, SPARC protein began to accumulate within the intersomitic clefts at the onset of trunk myotome contraction. The intersomitic enrichment of SPARC remained high as long as the myotomes remained electrically coupled, principally by gap junctions. As myotomes became innervated, SPARC expression decreased dramatically within the somites. SPARC was also found to be enriched within other tissues, such as the neural tube and epidermis. In addition, the selective spatial-temporal enrichment of SPARC suggests it makes important calcium-dependent contributions to early morphological development.

Mapping the initiation sites of in vitro transcripts of bacteriophage S13.

Analysis of in vitro run-off transcripts synthesized by Escherichia coli RNA polymerase holoenzyme on linearized bacteriophage S13 DNA templates revealed five major transcription initiation sites. The sites, located at positions 45, 982, 1823 (1827), 4876 and 5211, are each within the boundaries of promoters or putative promoters previously mapped by footprinting and RNA polymerase binding analyses. They correspond to initiations at promoters upstream of the A, B, and D genes, and at a medium-affinity and a high-affinity RNA polymerase binding site P5211, respectively. Sequence analysis of the 5'-ends of two transcripts confirmed their initiation with pppA at nt 982 and nt 5211, the B gene and high-affinity binding site P5211, respectively. Some of the transcripts initiated at nt 4876 and nt 5211 terminated at nt 64, providing direct evidence of the functionality of a p-independent termination site at nt 64.

Developmental anomalies of Xenopus embryos following microinjection of SPARC antibodies.

The function of SPARC (Secreted Protein, Acidic, Rich in Cysteine) in early embryonic development was assayed by microinjecting affinity-purified antibodies directed against SPARC into the blastocoel cavity of Xenopus embryos. Microinjection of SPARC antibodies did not appear to interfere with development until late neurulation. By hatching, a broad spectrum of external developmental anomalies were observable, including bent embryonic axes, accentuated ventral masses, shortened embryonic axes, and lack of visible eye pigment. Histological sections of injected embryos demonstrated that lack of visible eye pigmentation was often associated with deformities in eye development. Bending and shortening of the embryonic axis was associated with highly disorganized myotome patterns and loss of segmental boundaries. The results indicate a requirement for SPARC in the early morphological development of several tissues in Xenopus.

Cytoplasmic localization of the DNA virus frog erythrocytic virus.

In situ hybridization, using a biotinylated clone of frog erythrocytic virus (FEV), was conducted to determine the location of viral sequences in bullfrog erythrocytes. FEV-specific hybridization signals were found to correspond to mature cytoplasmic viral particles and assembly sites. These data are consistent with electron microscopic observations of viral assembly in the erythrocyte cytoplasm. Although FEV has morphological and biochemical properties similar to frog virus 3, our data suggest that the site of DNA replication and assembly of FEV is more similar to that of the poxviruses.

Molecular analysis of Xenopus laevis SPARC (Secreted Protein, Acidic, Rich in Cysteine). A highly conserved acidic calcium-binding extracellular-matrix protein.

SPARC (Secreted Protein, Acidic, Rich in Cysteine) is expressed as a 1.6 kb mRNA in Xenopus laevis. On the basis of cDNA sequence analysis, Xenopus SPARC has a core Mr of 32643, with one potential N-glycosylation site. Western analysis of SPARC isolated from Xenopus long bone indicates that the mature protein has an Mr of 43,000. At the amino acid level, Xenopus SPARC has 78-79% sequence similarity to mouse, bovine and human SPARC. The least-conserved region is found within the N-terminal glutamic acid-rich domain, with the C-terminal Ca(2+)-binding domain being the most conserved. Adult Xenopus tissues show the same pattern of tissue-specific distribution of SPARC mRNAs as adult mouse.

Expression of SPARC/osteonectin in tissues of bony and cartilaginous vertebrates.

To explore the biological functions of SPARC (secreted protein, acidic, rich in cysteine), a Ca(2+)-binding extracellular glycoprotein, we have examined its expression in an evolutionary diverse group of organisms. Similar patterns of SPARC mRNA expression were observed in adult mouse and rat tissues. SPARC transcripts represented 0.0002-0.0025% of the total RNA found in calvarium, lung, brain, and heart, whereas relatively low levels of SPARC RNA were detected in liver and kidney. Within nonmuscular tissues, a statistically significant correlation was observed between the tissue distribution of SPARC and cytoskeletal actin transcripts. Southern blot analysis revealed SPARC as a low or single-copy gene in an evolutionary diverse group of vertebrates. No hybridization signal was observed with the invertebrates examined. The tissue distribution of SPARC transcripts in the vertebrates examined was similar, except for sea lamprey and sea skate, two vertebrates that do not form mineralized bone. These data suggest that SPARC has multiple functions in mineralized and nonmineralized tissues of vertebrates.

Molecular analysis of cDNA coding for ZP3, a sperm binding protein of the mouse zona pellucida.

At fertilization, mammalian sperm bind is a species-specific manner to the extracellular zona pellucida that surrounds ovulated eggs. ZP3, an 83,000-85,000 Da glycoprotein of the murine zona pellucida, has been shown to inhibit sperm binding via its O-linked oligosaccharide side chains. We have recently isolated cDNA clones coding for ZP3 and have demonstrated that ZP3 transcripts are accumulated in oocytes where their expression is developmentally regulated during oogenesis. We now report that ZP3 mRNA is 1317 nt long with an estimated poly(A) tail of 200-300 nt. The short 29-nt 5' untranslated region is followed by a single open reading frame coding for a polypeptide chain of 46,307 Da which includes six possible sites for N-linked oligosaccharides. The N-terminus of ZP3 contains a potential 22-amino acid signal peptide which upon cleavage would result in a secreted core protein of 43,943 Da. The termination codon is a part of the AATAAA polyadenylation signal and is contained in an unusually short 16-nt 3' untranslated region. Sequences homologous to ZP3 are conserved among mammals and are expressed in ovarian tissue as mature transcripts with indistinguishable molecular weights.