Gamete recognition during fertilization in a red alga,Antithamnion nipponicum

Summary Fertilization in the marine red algaAntithamnion nipponicum is a highly specific process involving non-motile male gametes, spermatia, and female receptive structures, carpogonia. FITC-lectin and Calcofluor white ST labelling show that the outer cell walls of spermatia differ from vegetative cells in carbohydrate composition. Specific binding of the lectins to spermatial walls was confirmed by lectin-gold labelling on thin sections. Gametic recognition inAntithamnion nipponicum is based on the interaction of a surface carbohydrate on the spermatia with a surface carbohydrate receptor on the trichogynes. Spermatial binding to trichogynes is inhibited by pre-incubation with concanavalin A and trichogyne receptors are blocked by the complementary carbohydrate -D-methyl mannose. The inhibitory effects of concanavalin A to spermatial binding of trichogynes is reversed by preincubation with -D-methyl mannose. The combination of long spermatial appendages and a carbohydrate-carbohydrate receptor-based gamete recognition mechanism make fertilization in this species an efficient process.     

Gamete recognition in higher plants: an abstruse but charming mystery

Although much effort has been made to uncover the mechanism underlying double fertilization, little knowledge has been acquired for understanding the molecular base of gamete recognition, mainly because of technical limitations. Still,progress has been made in terms of the mechanism, including the identification of candidate molecules that are involved in gamete recognition in angiosperms. New cues for gamete recognition have been found by the successful separation of the gametes and construction of gamete-specific cDNA libraries in several species, and the application of molecular approaches for studying this process by mutations. Thus, the topic is considered an abstruse but charming mystery.     

The cell biology of fertilization: Gamete attachment and fusion

Fertilization is defined as the union of two gametes. During fertilization, sperm and egg fuse to form a diploid zygote to initiate prenatal development. In mammals, fertilization involves multiple ordered steps, including the acrosome reaction, zona pellucida penetration, sperm–egg attachment, and membrane fusion. Given the success of in vitro fertilization, one would think that the mechanisms of fertilization are understood; however, the precise details for many of the steps in fertilization remain a mystery. Recent studies using genetic knockout mouse models and structural biology are providing valuable insight into the molecular basis of sperm–egg attachment and fusion. Here, we review the cell biology of fertilization, specifically summarizing data from recent structural and functional studies that provide insights into the interactions involved in human gamete attachment and fusion.

IntroductionDuring sexual reproduction, the oocyte and sperm fuse to generate a new and unique embryo. The journey of a sperm to an egg ends in the ampulla of the female oviduct. From there, the sperm must overcome a number of physical and biochemical barriers. After undergoing the acrosome reaction and binding the ova, the sperm penetrates through the cumulus oophorus cells and the zona pellucida (ZP) to reach the perivitelline space (PVS) and oocyte membrane. Upon fusion of the sperm and egg membranes, the sperm nucleus and organelles are incorporated into the egg cytoplasm.An understanding of the mechanisms of mammalian fertilization is crucial to treat infertility and develop new methods of birth control. Infertility affects 15% of couples globally, and in one third of these cases, the underlying cause is unknown (Gelbaya et al., 2014). Developments in assisted reproductive technologies have provided couples with new options to conceive but may have epigenetic side effects (Mani et al., 2020). Furthermore, only 40% of couples manage to have a child despite 2 yr of treatment. Safety, efficacy, and acceptability of contraceptives are also critically important, but many current female contraceptive methods have side effects that limit long-term use (Aitken et al., 2008), while male contraceptives are limited to condoms or vasectomy (Kanakis and Goulis, 2015). A better understanding of the molecular players involved in fertilization is necessary to drive innovation in both assisted reproductive technologies and contraception.In this review, we will first briefly review the events that prepare the gametes for fertilization. We will then discuss how recent studies of genetically altered mice and structural biology efforts have shed light on the molecular mechanisms of sperm–egg attachment and fusion. We will also discuss the gaps in current knowledge and suggest new perspectives and future directions in the search for other protein factors involved at the gamete fusion synapse.Cell biology of gametesFertilization requires proper gametogenesis (oogenesis in the female and spermatogenesis in the male), which produces haploid cells and introduces diversity. Primordial germ cells (PGC) are the embryonic precursors to spermatocytes and ova. The cells produced by the first few divisions of the fertilized egg are totipotent and capable of differentiating into any cell type, including germ cells. PGCs originate within the primary ectoderm of the embryo and then migrate into the yolk sac. Between weeks 4 and 6, the PGCs migrate back into the posterior body wall of the embryo, where they stimulate cells of the adjacent coelomic epithelium and mesonephros to form primitive sex cords and induce the formation of the genital ridges and gonads. The sex (gonadal) cords surround the PGCs and give rise to the tissue that will nourish and regulate the development of the maturing sex cells (ovarian follicles in the female and Sertoli cells in the male).EggOogenesis is a complex differentiation process by which mature functional ova develop from germ cells (Fig. 1 A; Edson et al., 2009). In humans, oogenesis begins in the ovary at 6–8 wk of fetus development, when PGCs differentiate into oogonia. By the 12th week, several million oogonia enter prophase, the first meiotic division and become dormant until shortly before ovulation (Hayashi et al., 2020). Due to their large and watery nuclei, these cells are referred to as germinal vesicles (Pan and Li, 2019). These primary oocytes become enclosed by follicle cells to form primordial follicles. The number of primordial follicles peaks at ∼7 million by the fifth month of fetal life, with ∼700,000 left at birth and 400,000 by puberty (Marcozzi et al., 2018). All of the egg cells that the ovaries will release are already present at birth.Open in a separate windowFigure 1.Gametogenesis and fertilization. (A–C) Illustration of oogenesis and follicle development (A), spermatogenesis (B), and the major steps in fertilization (C): (1) initial contact, (2) acrosome reaction, (3) ZP penetration, (4) sperm–egg fusion, (5) entry of sperm nucleus, (6) cortical reaction, and (7) fusion of the sperm and egg nuclei. The oocyte with its ZP measures 130 μm in diameter. Created with BioRender.During each menstrual cycle, hormones from the hypothalamic–pituitary–gonadal axis restart the division of the primary oocytes in meiosis I and follicular development (Atwood and Vadakkadath Meethal, 2016). Primary follicles develop into secondary follicles, containing each growing oocyte surrounded by two or more layers of proliferating follicle cells. ZP glycoproteins are secreted by the oocyte of the primary follicle and possibly the follicular cells (Törmälä et al., 2008). Although these glycoproteins form a physical barrier between the follicle cells and the oocyte, follicle cells and the oocyte remain connected through transzonal cytoplasmic projections from the follicle cells until fertilization (Makabe et al., 2006). A reciprocal dialog between the oocyte and its surrounding follicular cells coordinates the different phases of follicular development and the maintenance of meiotic arrest (Dalbies-Tran et al., 2020). Oocyte-derived microvilli control female fertility by optimizing ovarian follicle selection in mice (Zhang et al., 2021). The epithelium of 5–12 primary follicles proliferates to form a multilayered capsule around the oocyte. A few of these growing follicles continue to enlarge in response to follicle-stimulating hormone (FSH; Visser and Themmen, 2014). A single follicle becomes dominant, and the others degenerate by atresia (Atwood and Vadakkadath Meethal, 2016). Meiosis of the oocyte in the mature preovulatory follicle is blocked until a surge in levels of FSH and luteinizing hormone that occurs midway through the menstrual cycle. The membrane of the germinal vesicle nucleus breaks down, the chromosomes align in metaphase, and the oocyte expels its first polar body. The secondary oocyte then begins the second meiotic division, which is arrested at the meiotic metaphase II stage until ovulation (Gougeon, 1996). Ovulation depends on the breakdown of the follicle wall and occurs ∼38 h after the increase in levels of FSH and luteinizing hormone (Holesh et al., 2021). The disruption of the follicle wall expulses the oocyte, which is captured by the fimbriated mouth of the oviduct and moved into the ampulla. The oocyte retains its ability to be fertilized for ∼24 h and completes meiosis only if it is fertilized.SpermIn contrast to oogenesis, which is complete before birth, spermatogenesis is a continuous process that begins at puberty (Fig. 1 B). In humans, spermatogenesis takes 74 d to complete; thus, multiple spermatogenesis events occur simultaneously to allow for continual sperm production. Spermatogenesis occurs in the testis in a stepwise manner, beginning with diploid spermatogonia at the basal surface of seminiferous tubules and ending with mature elongated spermatozoa that are released in tubule lumens in a process called spermiation (Clermont, 1972; Yang and Oatley, 2014). During spermatogenesis, mitosis results in gene amplification, meiosis results in genome reduction, and finally maturation occurs (Hess and Renato de Franca, 2008). At this stage, sperm are not motile and are fertilization incompetent. Two additional sperm maturational processes are required outside the testis. First, sperm undergo a maturation process during epididymal transit (Bedford et al., 1973) involving posttranslational modifications of previously synthesized proteins and acquisition of proteins from the epididymal epithelium (James et al., 2020; see text box). After ejaculation into the female reproductive tract, dilution triggers additional changes in sperm, collectively termed capacitation (see text box), that prepare the sperm for the acrosome reaction.Epididymal maturationSperm exchange with the epididymal epithelium occurs by direct interaction with epithelial cells, by interaction with soluble proteins in the epididymal fluid or via extracellular exosome-like vesicles released by epithelial cells called epididymosomes (James et al., 2020). The purposes of this exchange are to redistribute sperm proteins and change the composition and lipid balance of the sperm membrane. These changes take place during the transit from the epididymis initial segment, through the caput and the corpus, to the cauda where sperm are stored (Cornwall, 2009). Epididymal transit lasts 10–12 d in mammals, but storage is dependent on sexual activity. Since fertilization is not immediate, fertilizing capacities of the spermatozoa are preserved by decapacitation factors that are active in the epididymis. An example of a decapacitation factor is SPINK3, which is secreted by seminal vesicles; it impairs sperm membrane hyperpolarization and calcium influx through CatSper (Zalazar et al., 2020). Epididymal plasma and sperm represent only a small fraction (5%) of semen in men (Batruch et al., 2011). Two thirds of the volume of semen comes from the seminal vesicles and the other third from the prostate. These secretions protect the sperm and prevent early maturation.Sperm capacitationMore than 70 yr ago, Austin and Chang described capacitation as the changes required for sperm to fertilize oocytes in vivo (Austin, 1952; Chang, 1951). Once sperm enter the female reproductive tract, they undergo capacitation. Capacitation results in hyperactivation of sperm movement and initiation of the acrosome reaction (Saling et al., 1979; Florman and First, 1988). During capacitation, stabilizing or decapacitation factors that are adsorbed on the sperm plasma membrane are removed (Bedford and Chang, 1962). These agents that initiate removal of decapacitation factors are electrolytes, energy substrates, and proteins such as seminal plasma protein or albumin. Removal of decapacitation factors increases sperm plasma membrane fluidity, allowing an increase in the permeability to calcium, chloride, and bicarbonate ions (Gangwar and Atreja, 2015). Sperm motility depends on the membrane potential, intracellular pH, and balance of intracellular ions (reviewed in Nowicka-Bauer and Szymczak-Cendlak, 2021). The most important ion for this function is Ca²⁺ (Hwang et al., 2019). This secondary messenger is an important signaling pathways activator that regulates sperm motility (Finkelstein et al., 2020). The activation of soluble adenyl cyclases generates cyclic adenosine monophosphate that in turn activates serine/threonine protein kinase A, which induces a cascade of protein phosphorylation initiating the induction of sperm motility (Chen et al., 2000). Protein phosphorylation, sperm hyperactivation, and the acrosome reaction are used in vitro to evaluate capacitation. Capacitation can be induced in vitro by incubation in medium containing calcium, bicarbonate ions, and serum albumin (Touré, 2019).Mammalian sperm capacitation occurs during sperm migration in the female tract. Mammalian males ejaculate millions of sperm cells into the female reproductive tract, but only a few hundred sperm at most reach the oocytes. This massive elimination process likely prevents polyspermy (reviewed in Kölle, 2015). Selection of human sperm during the journey begins in the acidic environment of the vagina. In the cervix, only morphologically normal sperm can migrate. Some sperm immediately pass into the cervical mucus, whereas the remaining sperm becomes a part of the coagulum. The next selection occurs at the uterus–tubal junction, the connection between the uterus and the oviduct that represents a major obstacle for sperm migration (Kölle, 2015). Experiments in mice indicate that sperm motility alone is insufficient for sperm migration through the uterus–tubal junction (Fujihara et al., 2018). Uterine contractions facilitate sperm transport as do molecular interactions. Several proteins, such as ADAM3 and other ADAM family members, are known to be involved in this step in mice (Yamaguchi et al., 2009; Xiong et al., 2019); most ADAM proteins have human orthologues.Spermatogenesis takes place in a species-specific cycle called the seminiferous epithelial cycle and is regulated in particular through the hypothalamic–pituitary–testicular axis. Indeed, at puberty, the testes (interstitial steroidogenic Leydig cells) secrete an increased amount of testosterone, which triggers growth of the testes, maturation of the seminiferous tubules, and the commencement of spermatogenesis. The Sertoli cells are the major somatic cells present in the seminiferous tubules and are considered to be the main regulators of spermatogenesis. They orchestrate spermatogenesis by supporting spermatogonial stem cells, determining the testis size, organizing meiotic and postmeiotic development and sperm output, supporting androgen production by maintaining the development and function of Leydig cells, and regulating other aspects of testis function like peritubular myoid cells, immune cells, and the vasculature, which participate in the maintenance of the spermatogonial stem cell niche.Acrosome reactionThe acrosome is a secretory vesicle located on the anterior region of sperm that originates from the spermatid Golgi apparatus. An acrosomal granule is formed by the fusion of proacrosomal vesicles in the vicinity of the nucleus. The region increases in size and spreads over the anterior part of the nucleus. The acrosome reaction is driven by SNARE complexes and results in the exocytosis of the contents of the acrosome upon fusion of the plasma membrane with the outer acrosomal membrane (Fig. 1 C; reviewed in Okabe, 2016; De Blas et al., 2005). The timing of the acrosome reaction is critical. Only sperm that have undergone this reaction are fertilization competent, but when a high proportion of sperm undergo the acrosome reaction prematurely, success of in vitro fertilization is low (Wiser et al., 2014). Several studies indicate that only a fraction of sperm is capable of undergoing spontaneous acrosome reaction. In human and mice sperm samples, 15–20% of cells undergo spontaneous acrosome reaction (Nakanishi et al., 2001), whereas only 20–30% undergo progesterone-induced acrosome reaction (Stival et al., 2016), suggesting physiological heterogeneity of sperm population. In addition, Inoue et al. demonstrated that acrosome-reacted mouse spermatozoa recovered from the PVS can fertilize other eggs (Inoue et al., 2011).Based on in vitro data, it was thought that the acrosome reaction occurs when the sperm contacts the ZP, particularly the ZP3 protein (Litscher and Wassarman, 1996). Using transgenic mice that express fluorescent markers in the acrosome (Nakanishi et al., 1999) and the midpiece mitochondria (Hasuwa et al., 2010), real-time observation of acrosomal exocytosis was possible. These experiments showed that most mouse spermatozoa capable of fertilization had undergone the acrosome reaction before contact with the oocyte ZP (Jin et al., 2011). Most spermatozoa begin to react in the isthmus of the oviduct before reaching the ampulla (Hino et al., 2016; La Spina et al., 2016). Contact with the ZP in vitro probably makes it possible to complete a partial acrosome reaction. The most important function of the acrosome reaction is to induce changes in the sperm membrane (Okabe, 2016). The relocations of IZUMO1 and SPACA6, proteins essential for sperm–egg fusion, that occur after the acrosome reaction are illustrative examples of these changes (Sosnik et al., 2009; Barbaux et al., 2020; Satouh et al., 2012). The presence of these proteins on the sperm membrane, in addition to the classic markers Pisum sativum agglutinin, Peanut agglutinin lectins, or CD46, can be used as markers for the acrosome reaction (Ito and Toshimori, 2016). The acrosome and its disruption are both crucial for effective fertilization, as low fertilization rates are observed upon intracytoplasmic sperm injection of acrosome-intact sperm (Morozumi and Yanagimachi, 2005) or round spermatozoa lacking acrosomes (Dávila Garza and Patrizio, 2013).ZP penetrationThe ZP is a physical barrier between the oocyte and the follicular cells that forms from glycoproteins secreted from the primary follicles (Fig. 1 C). The human ZP consists of four glycoproteins (hZP1–hZP4; Harris et al., 1994). Mice, which have been used for most of the ZP studies in mammals, express only three ZP glycoproteins (mZP1–mZP3; Litscher and Wassarman, 2007). Analysis of mouse lines expressing human ZP proteins demonstrated that only hZP2 is important in human sperm–egg binding (Gupta, 2021). Experiments using purified native or recombinant human ZP proteins have shown that hZP1, hZP3, and hZP4 bind to the capacitated human spermatozoa and induce the acrosome reaction (Gupta, 2021). ZP1 is required for the structural integrity of the ZP (Chakravarty et al., 2008). To better understand the roles of ZP glycoproteins, further studies, particularly on ZP protein glycosylation, are needed. The species-specific binding of the ZP to sperm is presumably related to these carbohydrate moieties (Clark, 2014). The sialyl-Lewis(x) sequence is the major carbohydrate ligand for human sperm–egg binding (Pang et al., 2011). The current hypothesis that hZP1, hZP3, and hZP4 bind to capacitated sperm and hZP2 binds to sperm with intact acrosomes will need to be revisited due to the recent demonstration that the acrosome reaction takes place before ZP contact. Regardless, the role of the ZP in preventing polyspermy is clear. Indeed, ZP hardening is due to ZP2 cleavage by ovastacin, a protease released into the PVS by cortical granules after the first sperm–egg fusion (Burkart et al., 2012).Sperm–egg attachment and membrane fusionAfter penetration of the ZP, the sperm enters the PVS and can attach and fuse with the egg plasma membrane. The development of genetic knockout animal models has proven critical in determining the importance of various sperm and egg proteins in sperm–egg attachment and fusion. Surprisingly, genetic knockout studies revealed that many factors originally thought to be important for fertilization were in fact not necessary (reviewed in Okabe, 2018, 2015). The proteins from sperm and egg that are essential for sperm–egg membrane interaction and fusion are listed in

Bacterial antigens elicit T cell responses via adaptive and transitional immune recognition

T cells are a critical component of host immune responses against bacterial pathogens. T cell activation relies on recognition of antigen(s) derived from the bacteria, and this activation triggers potent biological effector mechanisms. Therefore, the characterization of antigens that are stimulatory for T cells provides insight into host-pathogen interactions and advances rational vaccine design. The adaptive immune response is defined by its ability to detect variable or unique single-gene products, whereas a 'transitional' immune system recognizes more conserved structures or products of multigene pathways. This transitional system functionally overlaps the canonical innate and adaptive immune responses. Antigen identification has relied upon biochemistry, genetics and expression cloning strategies. The development of computational approaches, fuelled by advances in immunology and genomic information, will facilitate the discovery of antigens and expand our understanding of both beneficial and pathological immune responses.     

Optimal immune responses: immunocompetence revisited

The function of the immune system of an animal is to provide defence against infection, in order to maximize fitness. Understanding this and, particularly, how limiting resources are traded off between costly immune responses and other physiological demands, is central to properly understanding life-history traits and their evolution. Here, we propose that functional (rather than immunological) measures of immune responses should be used when investigating this. We further suggest that optimal immune responses are context specific, rather than generic; that is, a maximum immune response is not necessarily optimal. The nature of an optimal immune response will depend on the specific circumstances and infection status of the animal. Identifying and understanding such optimality requires that the effects of different immune strategies on fitness be considered.     

Mannosylation in Candida albicans: role in cell wall function and immune recognition

The fungal cell wall is a dynamic organelle required for cell shape, protection against the environment and, in pathogenic species, recognition by the innate immune system. The outer layer of the cell wall is comprised of glycosylated mannoproteins with the majority of these post‐translational modifications being the addition of O‐ and N‐linked mannosides. These polysaccharides are exposed on the outer surface of the fungal cell wall and are, therefore, the first point of contact between the fungus and the host immune system. This review focuses on O‐ and N‐linked mannan biosynthesis in the fungal pathogen Candida albicans and highlights new insights gained from the characterization of mannosylation mutants into the role of these cell wall components in host–fungus interactions. In addition, we discuss the use of fungal mannan as a diagnostic marker of fungal disease.     

Pneumocystis: immune recognition and evasion

Pulmonary infection caused by the opportunistic fungal organism Pneumocystis continues to be a leading AIDS defining illness. The initiation of highly active antiretroviral therapy (HAART) in the HIV-infected population has led to a significant reduction in the incidence of Pneumocystis pneumonia (PCP), although recent trends suggest the incidence has plateaued rather than decreased. Host defense against Pneumocystis involves a delicate, concerted balance between the inflammatory response and immune-mediated clearance. Innate cellular immunity is a cornerstone in this response as it provides the initial recognition event that precipitates an immune response, ultimately leading to clearance of the organism from the host. This review will focus on carbohydrate moieties found in the Pneumocystis cell wall and the immune events that occur following their recognition.     

Gamete fusion site on the egg cell and autonomous establishment of cell polarity in the zygote

Gamete fusion activates the egg in animals and plants, and the gamete fusion site on the zygote might provide a possible cue for zygotic development and/or embryonic patterning. In angiosperms, a zygote generally divides into a two-celled proembryo consisting of an apical and a basal cell with different cell fates. This is a putative step in the formation of the apical-basal axis of the proembryo. We observed the positional relationship between the gamete fusion site and the division plane formed by zygotic cleavage using an in vitro fertilization system with rice gametes. There was no relationship between the gamete fusion site and the division plane leading to the two-celled proembryo. Thus, the gamete fusion site on the rice zygote does not appear to function as a determinant for positioning the zygote division plane, and the zygote apparently possesses autonomous potential to establish cell polarity along the apical-basal axis for its first cleavage.Key words: asymmetric division, egg cell, fertilization, gamete fusion, rice, sperm cell, two-celled proembryo, zygote     

DNA sensor cGAS-mediated immune recognition

The host takes use of pattern recognition receptors (PRRs) to defend against pathogen invasion or cellular damage. Among microorganism-associated molecular patterns detected by host PRRs, nucleic acids derived from bacteria or viruses are tightly supervised, providing a fundamental mechanism of host defense. Pathogenic DNAs are supposed to be detected by DNA sensors that induce the activation of NFκB or TBK1-IRF3 pathway. DNA sensor cGAS is widely expressed in innate immune cells and is a key sensor of invading DNAs in several cell types. cGAS binds to DNA, followed by a conformational change that allows the synthesis of cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) from adenosine triphosphate and guanosine triphosphate. cGAMP is a strong activator of STING that can activate IRF3 and subsequent type I interferon production. Here we describe recent progresses in DNA sensors especially cGAS in the innate immune responses against pathogenic DNAs.     

Human papillomavirus can escape immune recognition through Langerhans cell phosphoinositide 3-kinase activation

Human papillomavirus (HPV) infection of cervical epithelium is linked to the generation of cervical cancer. Although most women infected with HPV clear their lesions, the long latency period from infection to resolution indicates that HPV evolved immune escape mechanisms. Dendritic cells, which are targeted by vaccination procedures, incubated with HPV virus-like particles induce an HPV-specific immune response. Langerhans cells (LC), which are located at the sites of primary infection, do not induce a response implicating the targeting of LC as an immune escape mechanism used by HPV. LC incubated with HPV virus-like particles up-regulate the phosphoinositide 3-kinase (PI3-K) pathway and down-regulate MAPK pathways. With the inhibition of PI3-K and incubation with HPV virus-like particles, LC initiate a potent HPV-specific response. PI3-K activation in LC defines a novel escape mechanism used by HPV, and PI3-K inhibition may serve as an effective clinical target to enhance HPV immunity.     

Roles for glycosylation of cell surface receptors involved in cellular immune recognition.

The majority of cell surface receptors involved in antigen recognition by T cells and in the orchestration of the subsequent cell signalling events are glycoproteins. The length of a typical N-linked sugar is comparable with that of an immunoglobulin domain (30 A). Thus, by virtue of their size alone, oligosaccharides may be expected to play a significant role in the functions and properties of the cell surface proteins to which they are attached. A databank of oligosaccharide structures has been constructed from NMR and crystallographic data to aid in the interpretation of crystal structures of glycoproteins. As unambiguous electron density can usually only be assigned to the glycan cores, the remainder of the sugar is then modelled into the crystal lattice by superimposing the appropriate oligosaccharide from the database. This approach provides insights into the roles that glycosylation might play in cell surface receptors, by providing models that delineate potential close packing interactions on the cell surface. It has been proposed that the specific recognition of antigen by T cells results in the formation of an immunological synapse between the T cell and the antigen-presenting cell. The cell adhesion glycoproteins, such as CD2 and CD48, help to form a cell junction, providing a molecular spacer between opposing cells. The oligosaccharides located on the membrane proximal domains of CD2 and CD48 provide a scaffold to orient the binding faces, which leads to increased affinity. In the next step, recruitment of the peptide major histocompatibility complex (pMHC) by the T-cell receptors (TCRs) requires mobility on the membrane surface. The TCR sugars are located such that they could prevent non-specific aggregation. Importantly, the sugars limit the possible geometry and spacing of TCR/MHC clusters which precede cell signalling. We postulate that, in the final stage, the sugars could play a general role in controlling the assembly and stabilisation of the complexes in the synapse and in protecting them from proteolysis during prolonged T-cell engagement.     

Innate immune recognition of microbial cell wall components and microbial strategies to evade such recognitions

The innate immune system constitutes the first line of defence against invading microbes. The basis of this defence resides in the recognition of defined structural motifs of the microbes called “Microbial associated molecular patterns” that are absent in the host. Cell wall, the outer layer of both bacterial and fungal cells, a unique structure that is absent in the host and is recognized by the germ line encoded host receptors. Nucleotide oligomerization domain proteins, peptidoglycan recognition proteins and C-type lectins are host receptors that are involved in the recognition of bacterial cell wall (usually called peptidoglycan), whereas fungal cell wall components (N- and O-linked mannans, β-glucans etc.) are recognized by host receptors like C-type lectins (Dectin-1, Dectin-2, mannose receptor, DC-SIGN), Toll like receptors-2 and -4 (TLR-2 and TLR-4). These recognitions lead to activation of a variety of host signaling cascades and ultimate production of anti-microbial compounds including phospholipase A2, antimicrobial peptides, lysozyme, reactive oxygen and nitrogen species. These molecules act in cohort against the invading microbes to eradicate infections. Additionally pathogen recognition leads to the production of cytokines, which further activate the adaptive immune system. Both pathogenic and commensal bacteria and fungus use numerous strategies to subvert the host defence. These strategies include bacterial peptidoglycan glycan backbone modifications by O-acetylation, N-deacetylation, N-glycolylation and stem peptide modifications by amidation of meso-Diaminopimelic acid; fungal cell wall modifications by shielding the β-glucan layer with mannoproteins and α-1,3 glucan. This review focuses on the recent advances in understanding the role of bacterial and fungal cell wall in their innate immune recognition and evasion strategies.     

Macrophage recognition of immune complexes: development and application of novel cell surface labeling procedures

A fluorescein- and lactoperoxidase-conjugated ferritin-anti-ferritin immune complex has been prepared for cell surface labeling experiments on immune recognition and effector function. Lactoperoxidase (LPO) has been covalently coupled to affinity-purified anti-ferritin antibodies with p-benzoquinone by a modified version of the method of Ternynck and Avrameas [Ternynck, T., & Avrameas, S. (1976) Ann. Immunol. (Paris) 127C, 197]. The conjugate is a heterodimer of Mr230 000 with linkages to either or both of the heavy and light chains of the antibody, as judged by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence and presence of 2-mercaptoethanol. The conjugate retains antibody-binding activity as measured by a quantitative precipitin assay. When incorporated into immune complexes, the modified antibody also retains Fc receptor recognition ability as determined by erythrocyte-antibody rosette inhibition assays. Electron microscopy demonstrated that the antigen, ferritin, was monodisperse with complete apoprotein sheaths surrounding the core. Ferritin-anti-ferritin-LPO complexes were formed in 4-fold antigen excess. Complexes were verified by fluorescence and electron microscopy. Immune complexes were masked with "cold" iodine by use of the endogenous LPO activity. The complexes bound to cells at 4 degrees C as shown by electron microscopy and fluorescence video/intensification microscopy. The LPO delivered to the cell surface in this fashion can be utilized to iodinate the surface with 125I. Under saturation conditions, the labeling with local LPO delivery followed by SDS-PAGE and autoradiography is identical with labeling with free LPO. Labeling has also been conducted under conditions of substrate deficit.(ABSTRACT TRUNCATED AT 250 WORDS)     

昆虫免疫识别与病原物免疫逃避机理研究进展

昆虫在长期进化过程中形成复杂的天然免疫系统,病原识别是启动下游免疫反应的第一步,这一过程主要是由不同的模式识别蛋白来完成的。目前发现并鉴定的昆虫模式识别蛋白主要包括肽聚糖识别蛋白、类免疫球蛋白、β-1,3-葡聚糖结合蛋白、C型凝集素及具多功能的载脂蛋白等,不同的蛋白种类具有不同的结构、功能及识别对象。与昆虫免疫识别相对应的是,不同昆虫病原物在进化过程中发展出不同策略的免疫逃避能力,以战胜宿主免疫而致病或最终杀死昆虫。本文就昆虫免疫过程中不同模式识别蛋白的结合对象、结构与功能,以及逐渐兴起的病原物通过分子伪装等进行免疫逃避的研究进展进行了综述。并在此基础上,作者就昆虫免疫与昆虫病理研究的发展方向进行了展望,认为只有当两方面研究相结合时,才能更好地揭示昆虫宿主与病原物之间免疫与抗免疫的动态相互作用过程。     

Innate immune recognition of viral infection

Toll-like receptors (TLRs) are key molecules of the innate immune systems, which detect conserved structures found in a broad range of pathogens and triggers innate immune responses. A subset of TLRs recognize viral components and induce antiviral responses by producing type I interferons. Whereas TLR2 and TLR4 recognize viral components at the cell surface, TLR3, TLR7, TLR8 and TLR9 are exclusively expressed in endosomal compartments. After phagocytes internalize viruses or virus-infected apoptotic cells, viral nucleic acids are released in phagolysosomes and are recognized by these TLRs. Recent reports have shown that hosts also have a mechanism to detect replicating viruses in the cytoplasm in a TLR-independent manner. In this review, we focus on the viral recognition by innate immunity and the signaling pathways.     

The immune recognition of malaria antigens

Owing to the demonstration that the immune response of inbred mice to some defined malaria antigens is influenced by the major histocompatibility complex (MHC), and the finding that only a minority of individuals living in malaria-endemic areas appear to recognize such antigens, there are fears that synthetic subunit malaria vaccines will be poorly immunogenic in a substantial proportion of the target population. Such fears have been reinforced by the results of the first two human malaria vaccine trials. In this review Eleanor Riley, Olle Olerup and Marita Troye-Blomberg summarize the experimental evidence for MHC-related genetic restriction of malaria immunity and discuss some alternative explanations for nonresponsiveness in populations living in malaria-endemic areas.     

High-resolution structural insights into ligand binding and immune cell recognition by human lung surfactant protein D

Lung surfactant protein D (SP-D) can directly interact with carbohydrate residues on pulmonary pathogens and allergens, stimulate immune cells, and manipulate cytokine and chemokine profiles during the immune response in the lungs. Therapeutic administration of rfhSP-D, a recombinant homotrimeric fragment of human SP-D comprising the alpha-helical coiled-coil neck plus three CRDs, protects mice against lung allergy and infection caused by the fungal pathogen Aspergillus fumigatus. The high resolution crystal structures of maltose-bound rfhSP-D to 1.4A, and of rfhSP-D to 1.6A, define the fine detail of the mode and nature of carbohydrate recognition and provide insights into how a small fragment of human SP-D can bind to allergens/antigens or whole pathogens, and at the same time recruit and engage effector cells and molecules of humoral immunity. A previously unreported calcium ion, located on the trimeric axis in a pore at the bottom of the funnel formed by the three CRDs and close to the neck-CRD interface, is coordinated by a triad of glutamate residues which are, to some extent, neutralised by their interactions with a triad of exposed lysine residues in the funnel. The spatial relationship between the neck and the CRDs is maintained internally by these lysine residues, and externally by a glutamine, which forms a pair of hydrogen-bonds within an external cleft at each neck-CRD interface. Structural links between the central pore and the cleft suggest a possible effector mechanism for immune cell surface receptor binding in the presence of bound, extended natural lipopolysaccharide and phospholipid ligands. The structural requirements for such an effector mechanism, involving both the trimeric framework for multivalent ligand binding and recognition sites formed from more than one subunit, are present in both native hSP-D and rfhSP-D, providing a possible explanation for the significant biological activity of rfhSP-D.