N-glycan moieties of the crustacean egg yolk protein and their glycosylation sites

NK cells kill various cells using activating receptors, such as the natural cytotoxicity receptors (NCRs). NKp46 is a major NCR and is the only NCR expressed in mice (denoted Ncr1). Using Ncr1-deficient mice (Ncr1gfp/pfp) we demonstrated that Ncr1 controls various pathologies, and that in its absence Ncr1-related functions are impaired. In 2012, another Ncr1-related mouse was generated, named Noé, in which a random mutation, W32R, in position 32, impaired the Ncr1-Noé cell surface expression. Interestingly, in the Noé mice, Ncr1-dependent deficiencies were not observed. Additionally, the Noé-NK cells were hyperactivated, probably due to increased Helios expression, and the Noé mice demonstrate increased clearance of influenza and murine CMV. In contrast, in the Ncr1gfp/pfp mice infection with influenza was lethal and we show in the present study no difference in murine CMV infection between Ncr1gfp/pfp and wild-type (WT) mice. Because the foremost difference between the Noé and Ncr1gfp/gfp mice is the presence of a mutated Ncr1-Noé protein, we studied its properties. We show that Ncr1-Noé and various other Ncr1 mutants in position 32 can be expressed on the surface, albeit slowly and unstably, and that ligand recognition and function of the various Ncr1-Noé is similar to the WT Ncr1. We further show that the glycosylation pattern of Ncr1-Noé is aberrant, that the Ncr1-Noé proteins accumulate in the endoplasmic reticulum, and that the expression of Ncr1-Noé proteins, but not WT Ncr1, leads to increased Helios expression. Thus, we suggest that the NK hyperactivated phenotype observed in the Noé mice might result from the presence of the Ncr1-Noé protein.

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“…The Ncr1 Ig and Ncr1 W32R Ig N-linked glycans were released in solution with PNGase F as described by Roth et al (33).…”

Section: Preparation Of the Fusion Proteins For Sugar Content Analysimentioning

confidence: 99%

Expression, Function, and Molecular Properties of the Killer Receptor Ncr1-Noé

Glasner¹,

Šimić

Miklić

et al. 2015

The Journal of Immunology

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“…Sequence analysis of the various Vtg isoforms in zebrafish demonstrated the presence of several potential N-linked glycan sites within these C-terminal Vtg domains. Glycomic analyses of vitellogenin in other species have indicated that Vtg precursors typically bear high mannose-type N-glycans (24,25). To confirm the presence of high mannose N-glycans on zebrafish Vtg, we performed Coomassie stains and ConA lectin blots on control and Endo H-treated yolk lysates.…”

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confidence: 99%

Selective Yolk Deposition and Mannose Phosphorylation of Lysosomal Glycosidases in Zebrafish

Fan

Klein

Flanagan-Steet

et al. 2010

Journal of Biological Chemistry

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The regulation and function of lysosomal hydrolases during yolk consumption and embryogenesis in zebrafish are poorly understood. In an effort to better define the lysosomal biochemistry of this organism, we analyzed the developmental expression, biochemical properties, and function of several glycosidases in zebrafish eggs, embryos, and adult tissues. Our results demonstrated that the specific activity of most enzymes increases during embryogenesis, likely reflecting a greater need for turnover within the embryo as yolk-derived nutrients are depleted. Analysis of glycosidase activity in zebrafish and medaka eggs revealed selective deposition of enzymes required for the degradation of N-linked glycans, including an abundance of acidic mannosidases. Treatment of zebrafish embryos with the ␣-mannosidase inhibitor swainsonine resulted in the accumulation of glycosylated vitellogenin fragments and demonstrated a function for maternally deposited acid ␣-mannosidase in yolk consumption. Surprisingly, we also found that, unlike mammals, acid ␣-glucosidase from zebrafish and medaka does not appear to be modified with mannose 6-phosphate residues. We further showed these residues were not acquired on human acid ␣-glucosidase when expressed in zebrafish embryos, suggesting unique differences in the ability of the human and zebrafish N-acetylglucosamine-1-phosphotransferase to recognize and modify certain lysosomal glycosidases. Together, these results provide novel insight into the role of acidic glycosidases during yolk utilization and the evolution of the mannose 6-phosphate targeting system in vertebrates.

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“…Reduction, alkylation and trypsinization steps were carried out as described previously (Roth et al, 2010). The resulting peptides were loaded onto a home-made reverse-phase column (15cm long, 75μm internal diameter) packed with Jupiter C18, 300Å, 5μm beads (Phenomenex, Torrance, CA, USA) and connected to a Eksigent nano-LC system (Eksigent, Dublin, CA, USA).…”

Section: Mass Spectrometrymentioning

confidence: 99%

Hemocyanin with phenoloxidase activity in the chitin matrix of the crayfish gastrolith

Glazer

Tom

Weil

et al. 2013

Journal of Experimental Biology

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SUMMARYGastroliths are transient extracellular calcium deposits formed by the crayfish Cherax quadricarinatus von Martens on both sides of the stomach wall during pre-molt. Gastroliths are made of a rigid chitinous organic matrix, constructed as sclerotized chitin-protein microfibrils within which calcium carbonate is deposited. Although gastroliths share many characteristics with the exoskeleton, they are simpler in structure and relatively homogeneous in composition, making them an excellent cuticle-like model for the study of cuticular proteins. In searching for molt-related proteins involved in gastrolith formation, two integrated approaches were employed, namely the isolation and mass spectrometric analysis of proteins from the gastrolith matrix, and 454-sequencing of mRNAs from both the gastrolith-forming and sub-cuticular epithelia. SDS-PAGE separation of gastrolith proteins revealed a set of bands at apparent molecular masses of 75-85kDa; mass spectrometry data matched peptide sequences from the deduced amino acid sequences of seven hemocyanin transcripts. This assignment was then examined by immunoblot analysis using anti-hemocyanin antibodies, also used to determine the spatial distribution of the proteins in situ. Apart from contributing to oxygen transport, crustacean hemocyanins were previously suggested to be involved in several aspects of the molt cycle, including hardening of the new post-molt exoskeleton via phenoloxidation. The phenoloxidase activity of gastrolith hemocyanins was demonstrated. It was also noted that hemocyanin transcript expression during pre-molt was specific to the hepatopancreas. Our results thus reflect a set of functionally versatile proteins, expressed in a remote metabolic tissue and dispersed via the hemolymph to perform different roles in various organs and structures.

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N-glycan moieties of the crustacean egg yolk protein and their glycosylation sites

Cited by 34 publications

References 50 publications

Expression, Function, and Molecular Properties of the Killer Receptor Ncr1-Noé

Expression, Function, and Molecular Properties of the Killer Receptor Ncr1-Noé

Selective Yolk Deposition and Mannose Phosphorylation of Lysosomal Glycosidases in Zebrafish

Hemocyanin with phenoloxidase activity in the chitin matrix of the crayfish gastrolith

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