Joachim Bentrop

Molecular cloning of Drosophila Rh6 rhodopsin: the visual pigment of a subset of R8 photoreceptor cells 1

By screening retinal cDNA libraries for photoreceptor‐specifically expressed genes we have isolated and sequenced a cDNA clone encoding the rhodopsin (Rh6) of a subset of R8 photoreceptor cells of the Drosophila compound eye. Compared to the other visual pigments of Drosophila, this rhodopsin is equally homologous to Rh1 and Rh2 (51% amino acid identity) but shows only 32% and 33% amino acid identity with Rh3 and Rh4, respectively. The open reading frame codes for a protein of 369 amino acids (MW=41 691). The primary structure of Rh6 displays sites typical for rhodopsin molecules in general, for example, a chromophore binding site in transmembrane domain VII, sequence motifs in the intracellular loops 2 and 3 required for the binding of a heterotrimeric G‐protein, and a glycosylation site near the N‐terminus which seems to be important for protein transport and maturation. Since R8 cells are founder cells in the developing compound eye, the isolation of a rhodopsin gene expressed in these cells may aid the understanding of terminal differentiation of photoreceptor cells.

Glycan‐Specific Metabolic Oligosaccharide Engineering of C7‐Substituted Sialic Acids

Intact and integral glycosylation of membrane-associated as well as secreted glycoproteins has been shown to be essential for many aspects of the proper function of biological systems. Recombinantly expressed glycoproteins, such as antibodies, growth factors, hormones, vaccines, and contrast agents are key elements in medical applications. The quality of these therapeutically administered glycoproteins can be efficiently improved by the incorporation of chemically functionalized monosaccharides into their glycan moieties, a process denoted as metabolic oligosaccharide engineering (MOE). In addition to these pharmaceutical applications, MOE has greatly advanced diagnostics by localizing and visualizing glycans even in living animals. To date, a multitude of chemically modified monosaccharides have been designed for MOE applications. Owing to their terminal position at glycan structures of glycoproteins and relevance for cellular recognition, sialic acids and their metabolic precursor N-acetylmannosamine (ManNAc), are the most prominent targets for MOE. Several ManNAc derivatives with N-acetyl side-chain modifications have been synthesized and metabolically incorporated by the sialic acid biosynthetic pathway into a corresponding sialic acid C5 analogue (Figure 1). This approach was beneficial to extending the understanding of the biological role of the N-acyl side chain of sialic acids, for example, in virus infection or neuronal differentiation. Alternatively, C9 modifications of sialosides have also been achieved by directly administering synthetic sialic acid analogues. Additionally, selective cleavage of the glycol moiety led to a truncated sialic acid equipped glycans with an aldehyde for labeling reactions (Figure 1). All of these modifications address sialylation of both, Nand O-glycosylation of glycoproteins, to almost the same extent. Herein we investigate whether the biosynthetic machinery for sialic acids also tolerates other ManNAc derivatives as substrates, which are modified directly at the six-membered carbohydrate ring. The modification of the C4 position appeared most attractive, because it is not enzymatically modified during cellular glycoprotein production and would deliver previously unknown C7-modified sialic acid containing glycoproteins (Figure 1). To probe the biosynthetic promiscuity, we targeted a C4-modified ManNAc derivative, N-acetyl-4-azido-4-deoxymannosamine (4-azido-ManNAc, 1), in our study to enable postglycosylational conjugation and visualization by bioorthogonal reactions. N-acetyl-(1,3,6-O-acetyl)-4-azido-4-deoxy-mannosamine (Ac3-4-azido-ManNAc) was generated by an optimized literature method (Figure S1 in the Supporting InformaFigure 1. Methods for the structural modification of glycan-bound sialic acids by application of chemically modified ManNAc or direct periodate oxidation of glycan-bound sialic acids (left). Specific modification of the C7 position of sialic acids was achieved by C4-modified ManNAc in this study (right; note that to date these methods were carried individually, resulting in only one modification of a single sialic acid molecule).

Molecular evolution and expression of zebrafish St8SiaIII, an alpha-2,8-sialyltransferase involved in myotome development

Enzymes of the St8Sia family, a subgroup of the glycosyltransferases, mediate the transfer of sialic acid to glycoproteins or glycolipids. Here, we describe the cloning of the zebrafish St8SiaIII gene and study its developmental activity. A conserved synteny relationship among vertebrate chromosome regions containing St8SiaIII loci underscores an ancient duplication of this gene in the teleost fish lineage and a specific secondary loss of one paralog in the zebrafish. The single zebrafish St8SiaIII enzyme, which is expected to function as an oligosialyltransferase, lacks maternal activity, is weakly expressed during nervous system development, and shows a highly dynamic expression pattern in somites and somite‐derived structures. Morpholino knock‐down of St8SiaIII leads to anomalous somite morphologies, including defects in segment boundary formation and myotendious‐junction integrity. These phenotypes hint for a basic activity of zebrafish St8SiaIII during segmentation and somite formation, providing novel evidence for a non‐neuronal function of sialyltransferases during vertebrate development. Developmental Dynamics 237:808–818, 2008.

Ncam1a and Ncam1b: two carriers of polysialic acid with different functions in the developing zebrafish nervous system.

Polysialic acid (polySia) is mainly described as a glycan modification of the neural cell adhesion molecule NCAM1. PolySia-NCAM1 has multiple functions during the development of vertebrate nervous systems including axon extension and fasciculation. Phylogenetic analyses reveal the presence of two related gene clusters, NCAM1 and NCAM2, in tetrapods and fishes. Within the ncam1 cluster, teleost fishes express ncam1a (ncam) and ncam1b (pcam) as duplicated paralogs which arose from a second round of ray-finned fish-specific genome duplication. Tetrapods, in contrast, express a single NCAM1 gene. Using the zebrafish model, we identify Ncam1b as a novel major carrier of polySia in the nervous system. PolySia-Ncam1a is expressed predominantly in rostral regions of the developing nervous system, whereas polySia-Ncam1b prevails caudally. We show that ncam1a and ncam1b have different expression domains which only partially overlap. Furthermore, Ncam1a and Ncam1b and their polySia modifications serve different functions in axon guidance. Formation of the posterior commissure at the forebrain/midbrain junction requires polySia-Ncam1a on the axons for proper fasciculation, whereas Ncam1b, expressed by midbrain cell bodies, serves as an instructive guidance cue for the dorso-medially directed growth of axons. Spinal motor axons, on the other hand, depend on axonally expressed Ncam1b for correct growth toward their target region. Collectively, these findings suggest that the genome duplication in the teleost lineage has provided the basis for a functional diversification of polySia carriers in the nervous system.

Identification and Biochemical Characterization of Two Functional CMP-Sialic Acid Synthetases in Danio rerio

Background: Addition of sialic acid to the nonreducing end of glycoconjugates requires activation by CMP-sialic acid synthetase (CMAS). Results: In zebrafish, we identified two CMAS enzymes that differ in expression pattern, activities, and intracellular localization. Conclusion: Maintenance of two CMAS paralogues is attributed to subfunctionalization. Significance: Unraveling the individual functions of CMAS paralogues helps to elucidate the impact of sialylation in vertebrate development. Sialic acids (Sia) form the nonreducing end of the bulk of cell surface-expressed glycoconjugates. They are, therefore, major elements in intercellular communication processes. The addition of Sia to glycoconjugates requires metabolic activation to CMP-Sia, catalyzed by CMP-Sia synthetase (CMAS). This highly conserved enzyme is located in the cell nucleus in all vertebrates investigated to date, but its nuclear function remains elusive. Here, we describe the identification and characterization of two Cmas enzymes in Danio rerio (dreCmas), one of which is exclusively localized in the cytosol. We show that the two cmas genes most likely originated from the third whole genome duplication, which occurred at the base of teleost radiation. cmas paralogues were maintained in fishes of the Otocephala clade, whereas one copy got subsequently lost in Euteleostei (e.g. rainbow trout). In zebrafish, the two genes exhibited a distinct spatial expression pattern. The products of these genes (dreCmas1 and dreCmas2) diverged not only with respect to subcellular localization but also in substrate specificity. Nuclear dreCmas1 favored N-acetylneuraminic acid, whereas the cytosolic dreCmas2 showed highest affinity for 5-deamino-neuraminic acid. The subcellular localization was confirmed for the endogenous enzymes in fractionated zebrafish lysates. Nuclear entry of dreCmas1 was mediated by a bipartite nuclear localization signal, which seemed irrelevant for other enzymatic functions. With the current demonstration that in zebrafish two subfunctionalized cmas paralogues co-exist, we introduce a novel and unique model to detail the roles that CMAS has in the nucleus and in the sialylation pathways of animal cells.

UV‐light‐dependent binding of a visual arrestin 1 isoform to photoreceptor membranes in a neuropteran (Ascalaphus) compound eye

Arrestins are regulators of the active state of G‐protein‐coupled receptors. Towards elucidating the function of different arrestin subfamilies in sensory cells, we have isolated a novel arrestin 1, Am Arr1, from the UV photoreceptors of the neuropteran Ascalaphus macaronius. Am Arr1 forms a phylogenetic clade with antennal and visual Arr1 isoforms of invertebrates. Am Arr1 undergoes a light‐dependent binding cycle to photoreceptor membranes, as reported earlier only for members of the arrestin 2 subfamily. This suggests a common control mechanism for the active state of invertebrate rhodopsins and G‐protein‐coupled receptors of antennal sensory cells. Furthermore, it implies that a strict correlation of distinct arrestin isoforms to distinct functions is not a general principle for invertebrate sensory cells.

Drosophila Rhodopsin 7 can partially replace the structural role of Rhodopsin 1, but not its physiological function

Rhodopsin 7 (Rh7), a new invertebrate Rhodopsin gene, was discovered in the genome of Drosophila melanogaster in 2000 and thought to encode for a functional Rhodopsin protein. Indeed, Rh7 exhibits most hallmarks of the known Rhodopsins, except for the G-protein-activating QAKK motif in the third cytoplasmic loop that is absent in Rh7. Here, we show that Rh7 can partially substitute Rh1 in the outer receptor cells (R1–6) for rhabdomere maintenance, but that it cannot activate the phototransduction cascade in these cells. This speaks against a role of Rh7 as photopigment in R1–6, but does not exclude that it works in the inner photoreceptor cells.

Recessive Degeneration of Photoreceptor Cells Caused by Point Mutations in the Cytoplasmic Domains of Drosophila Rhodopsin

The cellular mechanism of photoreceptor cell death in inherited retinal degeneration is not yet understood. Mutations in photoreceptor-specifically expressed genes, for example the rhodopsin gene, have been identified as primary genetic defects. Using transgenic Drosophila as a model system, we investigated whether mutations in distinct amino acids which are conserved within the cytoplasmic domains throughout the rhodopsin family, namely Leu81, Asn86 or Glu271, may cause inherited retinal degeneration. Substitutions at these sites are shown to interfere with two rhodopsin functions: (i) Rhodopsin biosynthesis is partially blocked, leading to a lowered amount of functional rhodopsin in photoreceptor cells, (ii) Photoreceptor cells expressing mutant rhodopsins undergo age-dependent degeneration in a recessive manner. Degeneration starts with a deterioration of the membranes constituting the photoreceptive cell compartment. In later states of degeneration, photoreceptor cell bodies and the extracellular interphotoreceptor space are filled with remnants of the photoreceptive membrane. Retinal degeneration is interpreted to result from alterations in rhodopsin’s cytoplasmic surface which destabilize protein-protein interactions required in maintaining the architecture of the photoreceptive membrane compartment.