Mammalian glycosylation in immunity

Glycosylation produces a diverse and abundant repertoire of glycans, which are collectively known as the glycome. Glycans are one of the four fundamental macromolecular components of all cells, and are highly regulated in the immune system. Their diversity reflects their multiple biological functions that encompass ligands for proteinaceous receptors known as lectins. Since the discovery that selectins and their glycan ligands are important for the regulation of leukocyte trafficking, it has been shown that additional features of the vertebrate immune system are also controlled by endogenous cellular glycosylation. This Review focuses on the emerging immunological roles of the mammalian glycome.     

Chemical technologies for probing glycans

Glycans are central to many biological processes, but efforts to define their functions at the molecular level have been frustrated by a lack of suitable technologies. Here we highlight chemical tools that are beginning to address this need.     

Questioning the Ubiquity of Neofunctionalization

Gene duplication provides much of the raw material from which functional diversity evolves. Two evolutionary mechanisms have been proposed that generate functional diversity: neofunctionalization, the de novo acquisition of function by one duplicate, and subfunctionalization, the partitioning of ancestral functions between gene duplicates. With protein interactions as a surrogate for protein functions, evidence of prodigious neofunctionalization and subfunctionalization has been identified in analyses of empirical protein interactions and evolutionary models of protein interactions. However, we have identified three phenomena that have contributed to neofunctionalization being erroneously identified as a significant factor in protein interaction network evolution. First, self-interacting proteins are underreported in interaction data due to biological artifacts and design limitations in the two most common high-throughput protein interaction assays. Second, evolutionary inferences have been drawn from paralog analysis without consideration for concurrent and subsequent duplication events. Third, the theoretical model of prodigious neofunctionalization is unable to reproduce empirical network clustering and relies on untenable parameter requirements. In light of these findings, we believe that protein interaction evolution is more persuasively characterized by subfunctionalization and self-interactions.     

Coiled-Coil Proteins Facilitated the Functional Expansion of the Centrosome

Repurposing existing proteins for new cellular functions is recognized as a main mechanism of evolutionary innovation, but its role in organelle evolution is unclear. Here, we explore the mechanisms that led to the evolution of the centrosome, an ancestral eukaryotic organelle that expanded its functional repertoire through the course of evolution. We developed a refined sequence alignment technique that is more sensitive to coiled coil proteins, which are abundant in the centrosome. For proteins with high coiled-coil content, our algorithm identified 17% more reciprocal best hits than BLAST. Analyzing 108 eukaryotic genomes, we traced the evolutionary history of centrosome proteins. In order to assess how these proteins formed the centrosome and adopted new functions, we computationally emulated evolution by iteratively removing the most recently evolved proteins from the centrosomal protein interaction network. Coiled-coil proteins that first appeared in the animal–fungi ancestor act as scaffolds and recruit ancestral eukaryotic proteins such as kinases and phosphatases to the centrosome. This process created a signaling hub that is crucial for multicellular development. Our results demonstrate how ancient proteins can be co-opted to different cellular localizations, thereby becoming involved in novel functions.     

Protein glycosylation.

Glycan structures can modulate the biological properties and functions of glycoproteins. This has been shown by investigation of the biological activities and glycan structures of several recombinant glycoproteins. Glycan structures of glycoproteins differ according to the species and tissue producing them, and selection of an appropriate host-cell type can generate recombinant glycoproteins with new characteristics.     

The innovation triad: an EvoDevo agenda

This article introduces a special issue on evolutionary innovation and morphological novelty, two interrelated themes that have received a remarkable increase of attention over the past few years. We begin with a discussion of the question of whether innovation and novelty represent distinct evolutionary problems that require a distinct conceptualization. We argue that the mechanisms of innovation and their phenotypic results--novelty--can only be properly addressed if they are distinguished from the standard evolutionary themes of variation and adaptation, and we present arguments for making such a distinction. We propose that origination, the first formation of biological structures, is another distinct problem of morphological evolution, and that together with innovation and novelty it constitutes a conceptual complex we call the innovation triad. We define a problem agenda of the triad, which separates the analysis of the initiating conditions from the mechanistic realization of innovation, and we discuss the theoretical problems that arise from treating innovation as distinct from variation. Further, we categorize the empirical approaches that address themes of the innovation triad in recognizing four major strands of research: the morphology and systematics program, the gene regulation program, the epigenetic program, and the theoretical biology program. We provide examples of each program, giving priority to contributions in the present issue. In conclusion, we observe that the innovation triad is one of the defining topics of EvoDevo research and may represent its most pertinent contribution to evolutionary theory. We point out that an inclusion of developmental systems properties into evolutionary theory represents a shift of explanatory emphasis from the external factors of natural selection to the internal dynamics of developmental systems, complementing adaptation with emergence, and contingency with inherency.     

An end to endless forms: epistasis, phenotype distribution bias, and nonuniform evolution

Studies of the evolution of development characterize the way in which gene regulatory dynamics during ontogeny constructs and channels phenotypic variation. These studies have identified a number of evolutionary regularities: (1) phenotypes occupy only a small subspace of possible phenotypes, (2) the influence of mutation is not uniform and is often canalized, and (3) a great deal of morphological variation evolved early in the history of multicellular life. An important implication of these studies is that diversity is largely the outcome of the evolution of gene regulation rather than the emergence of new, structural genes. Using a simple model that considers a generic property of developmental maps-the interaction between multiple genetic elements and the nonlinearity of gene interaction in shaping phenotypic traits-we are able to recover many of these empirical regularities. We show that visible phenotypes represent only a small fraction of possibilities. Epistasis ensures that phenotypes are highly clustered in morphospace and that the most frequent phenotypes are the most similar. We perform phylogenetic analyses on an evolving, developmental model and find that species become more alike through time, whereas higher-level grades have a tendency to diverge. Ancestral phenotypes, produced by early developmental programs with a low level of gene interaction, are found to span a significantly greater volume of the total phenotypic space than derived taxa. We suggest that early and late evolution have a different character that we classify into micro- and macroevolutionary configurations. These findings complement the view of development as a key component in the production of endless forms and highlight the crucial role of development in constraining biotic diversity and evolutionary trajectories.     

Increased understanding of metabolism of secondary plant products frequently generates sketchy and incorrect statements concerning their evolution and function

Secondary plant products perform important functions within the complex interactions between plants and their environment, e.g. as protective agents against pathogens and herbivores, or as attractants for potential pollinators. We are all aware that the enormous diversity of these natural products resulted from evolutionary processes driven by the selection of advantageous properties. However, when these nexuses are mentioned, very often we incline to formulate ‘Plants have acquired the ability to synthesize secondary plant products in order to …’ without realising that such a statement contradicts the Darwinian principles of evolution and corresponds to a Lamarckian view of teleological evolution. One of the major reasons for these automatic and unconscious misapprehensions is because of the ambiguous usage of the term ‘biological function’, which is very often thought to comprise an intention or a special purpose.     

The impact of oxygen on metabolic evolution: a chemoinformatic investigation

The appearance of planetary oxygen likely transformed the chemical and biochemical makeup of life and probably triggered episodes of organismal diversification. Here we use chemoinformatic methods to explore the impact of the rise of oxygen on metabolic evolution. We undertake a comprehensive comparative analysis of structures, chemical properties and chemical reactions of anaerobic and aerobic metabolites. The results indicate that aerobic metabolism has expanded the structural and chemical space of metabolites considerably, including the appearance of 130 novel molecular scaffolds. The molecular functions of these metabolites are mainly associated with derived aspects of cellular life, such as signal transfer, defense against biotic factors, and protection of organisms from oxidation. Moreover, aerobic metabolites are more hydrophobic and rigid than anaerobic compounds, suggesting they are better fit to modulate membrane functions and to serve as transmembrane signaling factors. Since higher organisms depend largely on sophisticated membrane-enabled functions and intercellular signaling systems, the metabolic developments brought about by oxygen benefit the diversity of cellular makeup and the complexity of cellular organization as well. These findings enhance our understanding of the molecular link between oxygen and evolution. They also show the significance of chemoinformatics in addressing basic biological questions.     

Signals, pathways and splicing regulation

Alternative splicing of messenger RNA precursors is an extraordinary source of protein diversity and the regulation of this process is crucial for diverse cellular functions in both physiological and pathological situations. For many years, several signaling pathways have been implicated in alternative splicing regulation. Recent work has begun to unravel the molecular mechanisms by which extracellular stimuli activate signaling cascades that modulate the activity of the splicing machinery and therefore the splicing pattern of many different target messenger RNA precursors. These experiments are revealing unexpected aspects of the mechanism that control splicing and the consequences of the regulated splicing events. We summarize here the current knowledge about signal-induced alternative splicing regulation of Slo, NR1, CD44, CD45 and fibronectin genes, and also discuss the importance of some of these events in determination of cellular fate. Furthermore, we highlight the relevance of signal-induced changes in phosphorylation state and subcellular distribution of splicing factors as a way of regulating the splicing process. Lastly, we explore new and unexpected findings about regulated splicing in anucleated cells.     

The Biology of Prostaglandins and Related Eicosanoids in Invertebrates: Cellular, Organismal and Ecological Actions

SYNOPSIS. Prostaglandins and related eicosanoids are oxygenatedmetabolites of C20 polyunsaturated fatty acids. These compoundshave been detected in species representing all major animalphyla. The significance of eicosanoids lies in two broad areasof animal biology. In one, eicosanoids are involved in regulationof many cellular events. In the other, eicosanoids facilitatecertain ecological interactions. Eicosanoids are known bestin the narrow context of their clinical signif-icance in humanmedicine. In this essay we suggest a new, broader paradigm forunderstanding the meaning of eicosanoids. Under this paradigm,called the biological paradigm, we note eicosanoids were recruitedinto roles as biological signal moieties long before the originsof the Metazoa. During the ensuing evolutionary diversificationof animals, eicosanoids have been used in a vast diversity ofbiolog ical roles, some of which occur only in invertebrates.We think this diversity endows eicosanoids with unusual explanatorypower in apprehending biological phenomena. In this review,we recount the literature on eicosanoids in protozoans and procaryotes,then provide a detailed review of the roles of eicosanoids ininverte-brate immunity. We draw upon recent work in parasitoiogyto outline an ecological role of eicosanoids in host-parasiterelationships. It appears to us that eicosanoids exert profoundeffects at the cellular, organismal and ecological levels ofbiologicalorganization. We suggest that continued inquiry into the biologicalsignificance of eicosanoids will yield important new informationon invertebrates.     

Immune recruitment or suppression by glycan engineering of endogenous and therapeutic antibodies

Human serum IgG contains multiple glycoforms which exhibit a range of binding properties to effector molecules such as cellular Fc receptors. Emerging knowledge of how the Fc glycans contribute to the antibody structure and effector functions has opened new avenues for the exploitation of defined antibody glycoforms in the treatment of diseases. Here, we review the structure and activity of antibody glycoforms and highlight developments in antibody glycoengineering by both the manipulation of the cellular glycosylation machinery and by chemoenzymatic synthesis. We discuss wide ranging applications of antibody glycoengineering in the treatment of cancer, autoimmunity and inflammation. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

Evolutionary conservation and network structure characterize genes of phenotypic relevance for mitosis in human

The impact of gene silencing on cellular phenotypes is difficult to establish due to the complexity of interactions in the associated biological processes and pathways. A recent genome-wide RNA knock-down study both identified and phenotypically characterized a set of important genes for the cell cycle in HeLa cells. Here, we combine a molecular interaction network analysis, based on physical and functional protein interactions, in conjunction with evolutionary information, to elucidate the common biological and topological properties of these key genes. Our results show that these genes tend to be conserved with their corresponding protein interactions across several species and are key constituents of the evolutionary conserved molecular interaction network. Moreover, a group of bistable network motifs is found to be conserved within this network, which are likely to influence the network stability and therefore the robustness of cellular functioning. They form a cluster, which displays functional homogeneity and is significantly enriched in genes phenotypically relevant for mitosis. Additional results reveal a relationship between specific cellular processes and the phenotypic outcomes induced by gene silencing. This study introduces new ideas regarding the relationship between genotype and phenotype in the context of the cell cycle. We show that the analysis of molecular interaction networks can result in the identification of genes relevant to cellular processes, which is a promising avenue for future research.     

Glycobiomarkers by glycoproteomics and glycan profiling (glycomics): emergence of functionality

Glycans stand out from all classes of biomolecules because of their unsurpassed structural complexity. This is generated by variability in anomeric status of the glycosidic bond and its linkage points, ring size, potential for branching and introduction of diverse site-specific substitutions. What poses an enormous challenge for analytical processing is, at the same time, the basis for the fingerprint-like glycomic profiles of glycoconjugates and cells. What's more, the glycosylation machinery is sensitive to disease manifestations, earning glycan assembly a reputation as a promising candidate to identify new biomarkers. Backing this claim for a perspective in clinical practice are recent discoveries that even seemingly subtle changes in the glycan structure of glycoproteins, such as a N-glycan core substitution by a single sugar moiety, have far-reaching functional consequences. They are brought about by altering the interplay between the glycan and (i) its carrier protein and (ii) specific receptors (lectins). Glycan attachment thus endows the protein with a molecular switch and new recognition sites. Co-ordinated regulation of glycan display and presentation of the cognate lectin, e.g. in cancer growth regulation exerted by a tumour suppressor, further exemplifies the broad functional dimension inherent to the non-random shifts in glycosylation. Thus studies on glycobiomarkers converge with research on how distinct carbohydrate determinants are turned into bioactive signals.     

Evolutionary innovations and the organization of protein functions in genotype space

The organization of protein structures in protein genotype space is well studied. The same does not hold for protein functions, whose organization is important to understand how novel protein functions can arise through blind evolutionary searches of sequence space. In systems other than proteins, two organizational features of genotype space facilitate phenotypic innovation. The first is that genotypes with the same phenotype form vast and connected genotype networks. The second is that different neighborhoods in this space contain different novel phenotypes. We here characterize the organization of enzymatic functions in protein genotype space, using a data set of more than 30,000 proteins with known structure and function. We show that different neighborhoods of genotype space contain proteins with very different functions. This property both facilitates evolutionary innovation through exploration of a genotype network, and it constrains the evolution of novel phenotypes. The phenotypic diversity of different neighborhoods is caused by the fact that some functions can be carried out by multiple structures. We show that the space of protein functions is not homogeneous, and different genotype neighborhoods tend to contain a different spectrum of functions, whose diversity increases with increasing distance of these neighborhoods in sequence space. Whether a protein with a given function can evolve specific new functions is thus determined by the protein's location in sequence space.     

Cancer stem cell marker glycosylation: Nature,function and significance

Glycans are essential for the maintenance of normal biological function, with alterations in glycan expression being a hallmark of cancer. Cancer stem cells (CSCs) are a subset of cells within a tumour capable of self-renewal, cellular differentiation and resistances to conventional therapies. As is the case with stem cells, marker proteins present on the cell surface are frequently used to identify and enrich CSCs, with the expression of these markers statistical correlating with the likelihood of cancer recurrence and overall patient survival. As such CSC markers are of high clinical relevance. The majority of markers currently used to identify CSC populations are glycoproteins, and although the diverse biological roles for many of these markers are known, the nature and function of the glycan moiety on these glycoproteins remains to be fully elucidated. This mini-review summarises our current knowledge regarding the types and extent of CSC marker glycosylation, and the various roles that these glycans play in CSC biology, including in mediating cell adhesion, metastasis, evading apoptosis, tear shear resistance, tumour growth, maintaining pluripotency, self-renewal, trafficking, maintaining stability, maintaining enzymatic activity and aiding epithelial mesenchymal transitioning. Given that CSCs markers have multiple diverse biological functions, and are potentially of significant diagnostic and therapeutic benefit the search for new markers that are uniquely expressed on CSCs is vital to selectively target/identify this subset of cancer cells. As such we have also outlined how high-throughput lectin microarrays can be used to successfully profile the glycosylation status of CSC and to identify glyco-markers unique to CSCs.     

Mechanical Operation and Intersubunit Coordination of Ring-Shaped Molecular Motors: Insights from Single-Molecule Studies

Ring NTPases represent a large and diverse group of proteins that couple their nucleotide hydrolysis activity to a mechanical task involving force generation and some type of transport process in the cell. Because of their shape, these enzymes often operate as gates that separate distinct cellular compartments to control and regulate the passage of chemical species across them. In this manner, ions and small molecules are moved across membranes, biopolymer substrates are segregated between cells or moved into confined spaces, double-stranded nucleic acids are separated into single strands to provide access to the genetic information, and polypeptides are unfolded and processed for recycling. Here we review the recent advances in the characterization of these motors using single-molecule manipulation and detection approaches. We describe the various mechanisms by which ring motors convert chemical energy to mechanical force or torque and coordinate the activities of individual subunits that constitute the ring. We also examine how single-molecule studies have contributed to a better understanding of the structural elements involved in motor-substrate interaction, mechanochemical coupling, and intersubunit coordination. Finally, we discuss how these molecular motors tailor their operation—often through regulation by other cofactors—to suit their unique biological functions.     

Mechanical Operation and Intersubunit Coordination of Ring-Shaped Molecular Motors: Insights from Single-Molecule Studies

Ring NTPases represent a large and diverse group of proteins that couple their nucleotide hydrolysis activity to a mechanical task involving force generation and some type of transport process in the cell. Because of their shape, these enzymes often operate as gates that separate distinct cellular compartments to control and regulate the passage of chemical species across them. In this manner, ions and small molecules are moved across membranes, biopolymer substrates are segregated between cells or moved into confined spaces, double-stranded nucleic acids are separated into single strands to provide access to the genetic information, and polypeptides are unfolded and processed for recycling. Here we review the recent advances in the characterization of these motors using single-molecule manipulation and detection approaches. We describe the various mechanisms by which ring motors convert chemical energy to mechanical force or torque and coordinate the activities of individual subunits that constitute the ring. We also examine how single-molecule studies have contributed to a better understanding of the structural elements involved in motor-substrate interaction, mechanochemical coupling, and intersubunit coordination. Finally, we discuss how these molecular motors tailor their operation—often through regulation by other cofactors—to suit their unique biological functions.