Insight into the Regulation of Glycan Synthesis in Drosophila Chaoptin Based on Mass Spectrometry

BACKGROUND: A variety of N-glycans attached to protein are known to involve in many important biological functions. Endoplasmic reticulum (ER) and Golgi localized enzymes are responsible to this template-independent glycan synthesis resulting glycoforms at each asparagine residues. The regulation mechanism such glycan synthesis remains largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate the relationship between glycan structure and protein conformation, we analyzed a glycoprotein of Drosophila melanogaster, chaoptin (Chp), which is localized in photoreceptor cells and is bound to the cell membrane via a glycosylphosphatidylinositol anchor. Detailed analysis based on mass spectrometry revealed the presence of 13 N-glycosylation sites and the composition of the glycoform at each site. The synthetic pathway of glycans was speculated from the observed glycan structures and the composition at each N-glycosylation site, where the presence of novel routes were suggested. The distribution of glycoforms on a Chp polypeptide suggested that various processing enzymes act on the exterior of Chp in the Golgi apparatus, although virtually no enzyme can gain access to the interior of the horseshoe-shaped scaffold, hence explaining the presence of longer glycans within the interior. Furthermore, analysis of Chp from a mutant (RNAi against dolichyl-phosphate alpha-d-mannosyltransferase), which affects N-glycan synthesis in the ER, revealed that truncated glycan structures were processed. As a result, the distribution of glycoforms was affected for the high-mannose-type glycans only, whereas other types of glycans remained similar to those observed in the control and wild-type. CONCLUSIONS/SIGNIFICANCE: These results indicate that glycan processing depends largely on the backbone structure of the parent polypeptide. The information we obtained can be applied to other members of the LRR family of proteins

Genome-scale embryonic developmental profile of gene expression in the common house spider Parasteatoda tepidariorum

We performed RNA sequencing (RNA-Seq) at ten successive developmental stages in embryos of the common house spider Parasteatoda tepidariorum. Two independent datasets from two pairs of parents represent the normalized coverage of mapped RNA-Seq reads along scaffolds of the P. tepidariorum genome assembly. Transcript abundance was calculated against existing AUGUSTUS gene models. The datasets have been deposited in the Gene Expression Omnibus (GEO) Database at the National Center for Biotechnology Information (NCBI) under the accession number GSE112712

Additional file 12 of Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider

Additional file 12: Table S16. Primers and cDNA clones used for RNAi and in situ hybridization

Additional file 2 of Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider

Additional file 2: Table S1. List of DEG candidates identified by comparative transcriptome analysis of c versus i/p cells from stage-3 embryo (comparison I). Table S2. List of DEG candidates identified by comparative transcriptome analysis of p versus c/i cells from stage-3 embryo (comparison II). Table S3. List of DEG candidates identified by comparative transcriptome analysis of i versus c/p cells from stage-3 embryo (comparison III). Table S4. List of DEG candidates indentified by comparative transcriptome analysis of c versus p cells from stage-4 embryo. Table S5. List of DEG candidates identified by comparative transcriptome analysis of c versus p cells from early stage-5 embryo

Additional file 5 of Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider

Additional file 5: Movie S2. Time-lapse video showing phenotypes gfp, g26874, g7720, and g4238 pRNAi embryos. Time after egg laying (AEL) and time after start of recording are indicated. Movie starts are adjusted by the timing of blastoderm formation. Some of the embryos are the same as those shown in Fig. 3A. Scale bar, 100 µm

Additional file 14 of Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider

Additional file 14. Multi-fasta format file of 118 sequences (75 amino acid sites) used for the first ML analysis

Additional file 4 of Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider

Additional file 4: Table S6. Summary of a pilot pRNAi screen of 19 selected genes

Additional file 9 of Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider

Additional file 9: Table S9. List of DEGs identified by comparative transcriptome analysis of fuchi pRNAi versus untreated embryos at stage 2. Table S10. List of DEGs identified by comparative transcriptome analysis of fuchi pRNAi versus untreated embryos at stage 3. Table S11. List of DEGs identified by comparative transcriptome analysis of fuchi pRNAi versus untreated embryos at early stage 5