The relationship between ram sperm head morphometry and fertility depends on the procedures of acquisition and analysis used

Sperm head morphometry is a parameter in the evaluation of semen that has been associated with fertility in two ways: comparing morphometric measures between predefined groups of fertility; or analyzing morphometric data by multivariate techniques to identify cell populations. We analyzed the morphometry of ram sperm head by three procedures and checked its relationship with male fertility. A Computer-Aided Sperm Morphometric Assessment procedure (CASMA), an image analysis software (NIS-Elements) in combination with an optical microscope (MO-NIS) and this image analysis software in combination with a scanning electron microscope (SEM-NIS) were used. Eight morphometric parameters were assessed: length, width, area, perimeter, ellipticity, form factor, elongation and regularity. We observed significant differences between the morphometric data of sperm head obtained with three study procedures. The CASMA procedure shows the highest values for all parameters and the SEM-NIS procedure the lowest. The analysis of a semen sample, when only the mean of morphometric parameters is used to describe the cell population, is too limited to interpret their fertilizing capacity. It is essential to analyze the complex structure of the samples by defining subpopulations by multivariate methods. With few exceptions, the means of each morphometric parameter differ between the three subpopulations analyzed in each procedure. Only the subpopulations obtained with the MO-NIS procedure showed a significant correlation with male fertility. In short, it is necessary to establish an instrumental standard for the analysis of sperm morphometry to obtain reliable results and we believe that the MO-NIS system presents these basic requirements.     

The spermatogenesis and the sperm structure of Terebrantia (Thysanoptera, Insecta)

Spermatogenesis and the sperm structure of the terebrantian Aeolothrips intermedius Bagnall are described. Spermatogenesis consists of two mitotic divisions; the second is characterized by the loss of half of the spermatids, which have pyknotic nuclei. Early spermatids have two centrioles, but when spermiogenesis starts, a third centriole is produced. The three basal bodies give rise to three flagella; later these fuse into a single flagellum which contains three 9 + 0 axonemes. The basal bodies are surrounded by a large amount of centriole adjunct material. During spermiogenesis this material contributes to the shifting of the three axonemes towards the anterior sperm region parallel to the elongating nucleus, and it is transformed into a dense cylinder. In the mature spermatids the three axonemes amalgamate to create a bundle of 27 doublet microtubules. Near the end of spermiogenesis the dense cylinder of the centriole adjunct lies parallel to the nucleus and the axonemes. It ends where the mitochondrion appears at half-sperm length. We confirm that Terebrantia testes have a single sperm cyst; their sperm are characterized by a cylindrical nucleus, three axonemes fused into one, a small mitochondrion and a short cylindrical centriole adjunct which corresponds to the dense body described in a previous work. The acrosome is lacking. At the midpoint of the anterior half of the sperm the outline of the cross-section is bilobed, with the nucleus contained in a pocket evagination of the plasma membrane. These characters are discussed in light of a comparison between Tubulifera and Terebrantia.     

Drosophila tumor suppressor Merlin is essential for morphogenesis of mitochondria during sperm formation

Drosophila Merlin, an ortholog of the merlin protein encoded by the human Neurofibromatosis 2 (NF2) gene, is important for the regulation of cell proliferation and differentiation in the eye and wing. Also, it has been shown to be involved in male fertility in flies. In the present study, we formation using the comparative light and electron microscopic research of merlin mutants (mer3 and mer4) and ectopic expression of the Mer+ construct. Our work defines specific functions for Merlin in the mitochondria association and aggregation during the nebenkern formation and unfurling mitochondrial derivates during spermatid elongation. Possible role of Merlin as an adaptor protein that can link mitochondria with cytoskeleton is discussed.     

Disabled-2 is essential for endodermal cell positioning and structure formation during mouse embryogenesis

The signal transduction adapter protein Disabled-2 (Dab2) is one of the two mammalian orthologs of the Drosophila Disabled. The brain-specific Disabled-1 (Dab1) functions in positional organization of brain cells during development. Dab2 is widely distributed and is highly expressed in many epithelial cell types. The dab2 gene was interrupted by in-frame insertion of beta-galactosidase (LacZ) in embryonic stem cells and transgenic mice were produced. Dab2 expression was first observed in the primitive endoderm at E4.5, immediately following implantation. The homozygous Dab2-deficient mutant is embryonic lethal (earlier than E6.5) due to defective cell positioning and structure formation of the visceral endoderm. In E5.5 dab2 (-/-) conceptus, visceral endoderm-like cells are present in the deformed primitive egg cylinder; however, the visceral endoderm cells are not organized, the cells of the epiblast have not expanded, and the proamniotic cavity fails to form. Disorganization of the visceral endodermal layer is evident, as cells with positive visceral endoderm markers are scattered throughout the dab2 (-/-) conceptus. Only degenerated remains were observed at E6.5 for dab2 (-/-) embryos, and by E7.5, the defective embryos were completely reabsorbed. In blastocyst in vitro culture, initially cells with characteristics of endoderm, trophectoderm, and inner cell mass were observed in the outgrowth of the hatched dab2 (-/-) blastocysts. However, the dab2 (-/-) endodermal cells are much more dispersed and disorganized than those from wild-type blastocysts, the inner cell mass fails to expand, and the outgrowth degenerates by day 7. Thus, Dab2 is required for visceral endodermal cell organization during early mouse development. The absence of an organized visceral endoderm in Dab2-deficient conceptus leads to the growth failure of the inner cell mass. We suggest that Dab2 functions in a signal pathway to regulate endodermal cell organization using endocytosis of ligands from the blastocoel cavity as a positioning cue.     

Offspring production with cryopreserved sperm from a live-bearing fish Xiphophorus maculatus and implications for female fecundity

Xiphophorus fishes are well-established models for biomedical research of spontaneous or induced tumors, and their use in research dates back to the 1930s. Currently, 58 well-pedigreed lines exist among 24 Xiphophorus species housed as live animals at the Xiphophorus Genetic Stock Center. The technique of sperm cryopreservation has been applied to preserve these valuable genetic resources, and production of offspring has been reported with cryopreserved sperm in two species (X. helleri and X. couchianus). The goal of this research was to establish protocols for sperm cryopreservation and artificial insemination that yield live young in X. maculatus, a widely used research species. The objectives were to: 1) collect basic biological characteristics of males, and quantify the sperm production yield after crushing of dissected testis; 2) cryopreserve sperm from X. maculatus by adapting as necessary the protocols for sperm cryopreservation of X. helleri and X. couchianus; 3) use cryopreserved sperm to inseminate virgin females of X maculatus and other species (X. helleri and X. couchianus), and 4) compare experimental trials over a 3-year period to identify opportunities for improving female fecundity. In total, 117 males were used in this study with a standard length of 2.5 ± 0.3 cm (mean ± SD), body weight of 0.474 ± 0.149 g, and dissected testis weight of 7.1 ± 3.7 mg. Calculation of sperm availability showed 5.9 ± 2.8 × 10(6) sperm cells per mg of testis weight. Offspring were produced from cryopreserved sperm. Male-to-male variation (1-70%) was observed in post-thaw motility despite little variation in motility before freezing (60-90%) or genetic variation (~100 generations of sib-mating). Comparisons of biological factors of males did not have significant correlations with the production of live young, and the influence of females on production of young was identified from the comparison of artificial insemination over 3 years. Overall, this study describes offspring production from cryopreserved sperm in a third species of Xiphophorus fishes, and identifies the opportunities for improving female fecundity which is essential for establishment of germplasm repositories for Xiphophorus fishes.     

Cytoskeletal changes during generative cell division and sperm formation inTradescantia virginiana

Summary Cytoskeletal organization and chromosome behavior were studied inTradescantia generative cells prior to and during sperm formation using in vitro grown pollen tubes and fluorescence staining methods. Before pollen germination, the crescent-shaped generative cell contains a reticulate microtubule (Mt) system. The cell elongates dramatically after germination, and its Mts assume a helical to longitudinal arrangement. Chromosome condensation is evident approximately 3hr after germination. Kinetochores appear as dark interruptions in the Mt array, and thus seem to attach directly to interphase fibers. No metaphase plate typical of other cells is observed with either DAPI or anti-tubulin staining. Instead, the chromosomes adopt a twisted or braided arrangement, with kinetochores distributed along the length of the cell and kinetochore fibers linked to each other and to surrounding fibers. Anaphase is characterized by a staggered, overlapping separation of chromosomes and by elongation of Mt branches connecting opposing kinetochore fibers. Cytokinesis appears to utilize a furrowing process; a phragmoplast or cell plate was never seen. As a result of these events, the sperm directly inherit their cytoskeleton from generative cell Mts involved in division. No actin fibers are observed at any stage using rhodamine-phalloidin staining. The results are discussed in terms of other reports on sperm formation, possible mitotic and cytokinetic mechanisms, and past distinctions between Mt arrays in higher plant somatic cells.Abbreviations CD cytochalasin D - DAPI 46-diamidino-2-phenyl-indole - DMSO dimethylsulfoxide - K-fiber kinetochore fiber - Mf microfilament - Mt microtubule - PPB preprophase Mt band - RP rhodamine phalloidin     

Cul4A is essential for spermatogenesis and male fertility

The mammalian Cul4 genes, Cul4A and Cul4B, encode the scaffold components of the cullin-based E3 ubiquitin ligases. The two Cul4 genes are functionally redundant. Recent study indicated that mice expressing a truncated CUL4A that fails to interact with its functional partner ROC1 exhibit no developmental phenotype. We generated a Cul4A−/− strain lacking exons 4–8 that does not express any detectable truncated protein. In this strain, the male mice are infertile and exhibit severe deficiencies in spermatogenesis. The primary spermatocytes are deficient in progression through late prophase I, a time point when expression of the X-linked Cul4B gene is silenced due to meiotic sex chromosome inactivation. Testes of the Cul4A−/− mice exhibit extensive apoptosis. Interestingly, the pachytene spermatocytes exhibit persistent double stranded breaks, suggesting a deficiency in homologous recombination. Also, we find that CUL4A localizes to the double stranded breaks generated in pre-pachytene spermatocytes. The observations identify a novel function of CUL4A in meiotic recombination and demonstrate an essential role of CUL4A in spermatogenesis.     

The postacrosomal assembly of sperm head protein, PAWP, is independent of acrosome formation and dependent on microtubular manchette transport

PAWP (postacrosomal sheath WW domain-binding protein) exclusively resides in the postacrosomal sheath (PAS) of the sperm perinuclear theca (PT). Because of the importance of this region in initiating oocyte activation during mammalian fertilization [Sutovsky, P., Manandhar, G., Wu, A., Oko, R., 2003. Interactions of sperm perinuclear theca with the oocyte: implications for oocyte activation, anti-polyspermy defense, and assisted reproduction. Microsc. Res. Tech. 61, 362-378; Wu, A., Sutovsky, P., Manandhar, G., Xu, W., Katayama, M., Day, B.N., Park, K.W., Yi, Y.J., Xi, Y.W., Prather, R.S., Oko, R., 2007. PAWP, A sperm specific ww-domain binding protein, promotes meiotic resumption and pronuclear development during fertilization. J. Biol. Chem. 282, 12164-12175], we were interested in resolving the origin and assembly of its proteins during spermatogenesis, utilizing PAWP as a model. Based on previous PT developmental studies, we predicted that the assembly of PAWP is dependent on microtubule-manchette protein transport and manchette descent and independent of subacrosomal PT formation. Consequently, we hypothesized that PAWP will colocalize with manchette microtubules during spermiogenesis. Utilizing specific antibodies, PAWP was first detected in the cytoplasmic lobe of spermatids beginning to undergo elongation and became most prominent in this region just prior to and during manchette descent. During this peak period, PAWP was concentrated over the manchette and colocalized with alpha- and beta-tubulin. It was then assembled as part of the PAS in the wake of manchette descent over the caudal half of the elongated spermatid nucleus. PAWP mRNA, on the other hand, was first detected in mid-pachytene spermatocytes, peaked by early round spermatids, and declined during spermatid elongation. In order to confirm that PAWP-PAS assembly was independent of subacrosomal PT development, PAWP immunolocalization was performed on the testes of NB-DNJ-treated mice which fail to form an acrosome and subacrosomal layer during spermiogenesis [van der Spoel, A.C., Jeyakumar, M., Butters, T.D., Charlton, H.M., Moore, H.D., Dwek, R.A., Platt, F.M., 2002. Reversible infertility in male mice after oral administration of alkylated imino sugars: a nonhormonal approach to male contraception. Proc. Natl. Acad. Sci. U.S.A. 99, 17173-17178] but whose elongated spermatids still retain egg-activating ability [Suganuma, R., Walden, C.M., Butters, T.D., Platt, F.M., Dwek, R.A., Yanagimachi, R., and van der Spoel, A.C., 2005. Alkylated imino sugars, reversible male infertility-inducing agents, do not affect the genetic integrity of male mouse germ cells during short-term treatment despite induction of sperm deformities. Biol. Reprod. 72, 805-813]. The same temporal and manchette-based pattern of PAWP-PAS assembly during spermiogenesis was evident as in controls supporting our hypothesis that PAS assembly is independent of subacrosomal PT formation and that egg-activating ability resides within the PAS.     

Aquaporin3 is a sperm water channel essential for postcopulatory sperm osmoadaptation and migration

In the journey from the male to female reproductive tract, mammalian sperm experience a natural osmotic decrease (e.g., in mouse, from ~415 mOsm in the cauda epididymis to ~310 mOsm in the uterine cavity). Sperm have evolved to utilize this hypotonic exposure for motility activation, meanwhile efficiently silence the negative impact of hypotonic cell swelling. Previous physiological and pharmacological studies have shown that ion channel-controlled water influx/efflux is actively involved in the process of sperm volume regulation; however, no specific sperm proteins have been found responsible for this rapid osmoadaptation. Here, we report that aquaporin3 (AQP3) is a sperm water channel in mice and humans. Aqp3-deficient sperm show normal motility activation in response to hypotonicity but display increased vulnerability to hypotonic cell swelling, characterized by increased tail bending after entering uterus. The sperm defect is a result of impaired sperm volume regulation and progressive cell swelling in response to physiological hypotonic stress during male-female reproductive tract transition. Time-lapse imaging revealed that the cell volume expansion begins at cytoplasmic droplet, forcing the tail to angulate and form a hairpin-like structure due to mechanical membrane stretch. The tail deformation hampered sperm migration into oviduct, resulting in impaired fertilization and reduced male fertility. These data suggest AQP3 as an essential membrane pathway for sperm regulatory volume decrease (RVD) that balances the "trade-off" between sperm motility and cell swelling upon physiological hypotonicity, thereby optimizing postcopulatory sperm behavior.     

Soluble adenylyl cyclase (sAC) is indispensable for sperm function and fertilization

We previously demonstrated that male mice deficient in the soluble adenylyl cyclase (sAC) are sterile and produce spermatozoa with deficits in progressive motility and are unable to fertilize zona-intact eggs. Here, analyses of sAC(-/-) spermatozoa provide additional insights into the functions linked to cAMP signaling. Adenylyl cyclase activity and cAMP content are greatly diminished in crude preparations of sAC(-/-) spermatozoa and are undetectable after sperm purification. HCO(3)(-) is unable to rapidly accelerate the flagellar beat or facilitate evoked Ca(2+) entry into sAC(-/-) spermatozoa. Moreover, the delayed HCO(3)(-)-dependent increases in protein tyrosine phosphorylation and hyperactivated motility, which occur late in capacitation of wild-type spermatozoa, do not develop in sAC(-/-) spermatozoa. However, sAC(-/-) sperm fertilize zona-free oocytes, indicating that gamete fusion does not require sAC. Although ATP levels are significantly reduced in sAC(-/-) sperm, cAMP-AM ester increases flagellar beat frequency, progressive motility, and alters the pattern of tyrosine phosphorylated proteins. These results indicate that sAC and cAMP coordinate cellular energy balance in wild-type sperm and that the ATP generating machinery is not operating normally in sAC(-/-) spermatozoa. These findings demonstrate that sAC plays a critical role in cAMP signaling in spermatozoa and that defective cAMP production prevents engagement of multiple components of capacitation resulting in male infertility.     

Mammalian actin binding protein 1 is essential for endocytosis but not lamellipodia formation: functional analysis by RNA interference

Mammalian actin binding protein 1 (mAbp1, also called SH3P7/Hip55) is structurally and functionally related to yeast Abp1 and to cortactin, both of which have been implicated in endocytotic processes. mAbp1 associates through its SH3 domain with dynamin, a large GTPase essential for vesicle fission. To clarify the function of mAbp1, we specifically knocked down its expression in human embryonic kidney 293T cells, using RNA interference (RNAi). Co-transfection of a short interfering RNA (siRNA) together with a plasmid coding for a surface marker, followed by purification of transfected cells, enabled us to obtain a cell population having up to 90% inhibition of mAbp1 expression. In mAbp1-knocked down cells, transferrin (Tf) receptor endocytosis was significantly inhibited and intracellular distribution of the early endosomal compartment was modified. In contrast, in these cells actin and microtubule filaments appeared normal, and formation of lamellipodia induced by active Rac was not inhibited. This study provides definitive evidence that mAbp1 is indispensable for receptor-mediated endocytosis.     

Tdrkh is essential for spermatogenesis and participates in primary piRNA biogenesis in the germline

Piwi proteins and Piwi‐interacting RNAs (piRNAs) repress transposition, regulate translation, and guide epigenetic programming in the germline. Here, we show that an evolutionarily conserved Tudor and KH domain‐containing protein, Tdrkh (a.k.a. Tdrd2), is required for spermatogenesis and involved in piRNA biogenesis. Tdrkh partners with Miwi and Miwi2 via symmetrically dimethylated arginine residues in Miwi and Miwi2. Tdrkh is a mitochondrial protein often juxtaposed to pi‐bodies and piP‐bodies and is required for Tdrd1 cytoplasmic localization and Miwi2 nuclear localization. Tdrkh mutants display meiotic arrest at the zygotene stage, attenuate methylation of Line1 DNA, and upregulate Line1 RNA and protein, without inducing apoptosis. Furthermore, Tdrkh mutants have severely reduced levels of mature piRNAs but accumulate a distinct population of 1′U‐containing, 2′O‐methylated 31–37 nt RNAs that largely complement the missing mature piRNAs. Our results demonstrate that the primary piRNA biogenesis pathway involves 3′→5′ processing of 31–37 nt intermediates and that Tdrkh promotes this final step of piRNA biogenesis but not the ping‐pong cycle. These results shed light on mechanisms underlying primary piRNA biogenesis, an area in which information is conspicuously absent.     

Sperm head morphometry in ejaculates of adult marmosets (Callithrix jacchus): A model for studying sperm subpopulations and among-donor variations

In humans and other mammals, sperm morphology has been considered one of the most important predictive parameters of fertility. The objective was to determine the presence and distribution of sperm head morphometric subpopulations in a nonhuman primate model (Callithrix jacchus), using an objective computer analysis system and principal component analysis (PCA) methods to establish the relationship between the subpopulation distribution observed and among-donor variation. The PCA method revealed a stable number of principal components in all donors studied, that represented more than 85% of the cumulative variance in all cases. After cluster analysis, a variable number (from three to seven) sperm morphometric subpopulations were identified with defined sperm dimensions and shapes. There were differences in the distribution of the sperm morphometric subpopulations (P < 0.001) in all ejaculates among the four donors analyzed. In conclusion, in this study, computerized sperm analysis methods combined with PCA cluster analyses were useful to identify, classify, and characterize various head sperm morphometric subpopulations in nonhuman primates, yielding considerable biological information. In addition, because all individuals were kept in the same conditions, differences in the distribution of these subpopulations were not attributed to external or management factors. Finally, the substantial information derived from subpopulation analyses provided new and relevant biological knowledge which may have a practical use for future studies in human and nonhuman primate ejaculates, including identifying individuals more suitable for assisted reproductive technologies.     

Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice

Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leads to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor^flox/flox mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function.     

Drosophila paramyosin is important for myoblast fusion and essential for myofibril formation

Paramyosin is a major structural protein of thick filaments in invertebrate muscles. Coiled-coil dimers of paramyosin form a paracrystalline core of these filaments, and the motor protein myosin is arranged on the core surface. To investigate the function of paramyosin in myofibril assembly and muscle contraction, we functionally disrupted the Drosophila melanogaster paramyosin gene by mobilizing a P element located in its promoter region. Homozygous paramyosin mutants die at the late embryo stage. Mutants display defects in both myoblast fusion and in myofibril assembly in embryonic body wall muscles. Mutant embryos have an abnormal body wall muscle fiber pattern arising from defects in myoblast fusion. In addition, sarcomeric units do not assemble properly and muscle contractility is impaired. We confirmed that these defects are paramyosin-specific by rescuing the homozygous paramyosin mutant to adulthood with a paramyosin transgene. Antibody analysis of normal embryos demonstrated that paramyosin accumulates as a cytoplasmic protein in early embryo development before assembling into thick filaments. We conclude that paramyosin plays an unexpected role in myoblast fusion and is important for myofibril assembly and muscle contraction.     

The small heat shock protein ODF1/HSPB10 is essential for tight linkage of sperm head to tail and male fertility in mice

Sperm motility and hence male fertility strictly depends on proper development of the sperm tail and its tight anchorage to the head. The main protein of sperm tail outer dense fibers, ODF1/HSPB10, belongs to the family of small heat shock proteins that function as molecular chaperones. However, the impact of ODF1 on sperm tail formation and motility and on male fecundity is unknown. We therefore generated mutant mice in which the Odf1 gene was disrupted. Heterozygous mutant male mice are fertile while sperm motility is reduced, but Odf1-deficient male mice are infertile due to the detachment of the sperm head. Although headless tails are somehow motile, transmission electron microscopy revealed disturbed organization of the mitochondrial sheath, as well as of the outer dense fibers. Our results thus suggest that ODF1, besides being involved in the correct arrangement of mitochondrial sheath and outer dense fibers, is essential for rigid junction of sperm head and tail. Loss of function of ODF1, therefore, might account for some of the cases of human infertility with decapitated sperm heads. In addition, since sperm motility is already affected in heterozygous mice, impairment of ODF1 might even account for some cases of reduced fertility in male patients.     

CCN2/connective tissue growth factor is essential for pericyte adhesion and endothelial basement membrane formation during angiogenesis

CCN2/Connective Tissue Growth Factor (CTGF) is a matricellular protein that regulates cell adhesion, migration, and survival. CCN2 is best known for its ability to promote fibrosis by mediating the ability of transforming growth factor β (TGFβ) to induce excess extracellular matrix production. In addition to its role in pathological processes, CCN2 is required for chondrogenesis. CCN2 is also highly expressed during development in endothelial cells, suggesting a role in angiogenesis. The potential role of CCN2 in angiogenesis is unclear, however, as both pro- and anti-angiogenic effects have been reported. Here, through analysis of Ccn2-deficient mice, we show that CCN2 is required for stable association and retention of pericytes by endothelial cells. PDGF signaling and the establishment of the endothelial basement membrane are required for pericytes recruitment and retention. CCN2 induced PDGF-B expression in endothelial cells, and potentiated PDGF-B-mediated Akt signaling in mural (vascular smooth muscle/pericyte) cells. In addition, CCN2 induced the production of endothelial basement membrane components in vitro, and was required for their expression in vivo. Overall, these results highlight CCN2 as an essential mediator of vascular remodeling by regulating endothelial-pericyte interactions. Although most studies of CCN2 function have focused on effects of CCN2 overexpression on the interstitial extracellular matrix, the results presented here show that CCN2 is required for the normal production of vascular basement membranes.     

The oligomeric structure of vaccinia viral envelope protein A27L is essential for binding to heparin and heparan sulfates on cell surfaces: a structural and functional approach using site-specific mutagenesis

The soluble domain of the self-assembly vaccinia virus envelope protein A27L, sA27L-aa, consists of a flexible extended coil at the N terminus and a rigid hydrophobic coiled-coil region at the C terminus. In the former, a basic strip of 12 residues is responsible for binding to cell-surface heparan sulfates. Although the latter is believed to mediate self-assembly, its biological role is unclear. However, an in vitro bioassay showed that peptides comprising the 12 residue basic region alone failed to interact with heparin, suggesting that the C-terminal coiled-coil region might serve an indispensable role in biological function. To explore this structural and functional relationship, we performed site-specific mutagenesis in an attempt to specifically disrupt the hydrophobic core of the coiled coil. Three single mutants, L47A, L51A, and L54A, and one triple mutant, L47,51,54A, were expressed and purified from Escherichia coli. The physical properties of the mutants were carefully examined by gel-filtration chromatography, CD, and NMR spectroscopy, and the biological activities were assessed by an in vitro SPR bioassay and three in vivo bioassays: binding to cells, blocking virus infection and blocking cell fusion. We showed that the L47A mutant, which is similar to the parental sA27L-aa in forming a hexamer, is biologically active. L51A and L54A mutants form tetramers and are less active. Notably, in the triple mutant, the self-assembly hydrophobic core structure is uncoiled; as a consequence, the tetrameric structure is biologically inactive. Thus, we conclude that the leucine residues, in particular Leu51 and Leu54, sustain the hydrophobic core structure that is essential for the biological function of vaccinia virus envelope protein A27L, binding to cell-surface heparan sulfate.