scholarly journals Bombyx mori β1,4-N-acetylgalactosaminyltransferase possesses relaxed donor substrate specificity in N-glycan synthesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hiroyuki Kajiura ◽  
Ryousuke Miyauchi ◽  
Akemi Kakudo ◽  
Takao Ohashi ◽  
Ryo Misaki ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Developmental Stage ◽  
Bombyx Mori ◽  
Insect Cells ◽  
Glucuronic Acid ◽  
Silk Gland ◽  
Eukaryotic Cells ◽  
Sf9 Cells ◽  
Glcnac Residue ◽  
Donor Substrate

AbstractN-Glycosylation is one of the most important post-translational protein modifications in eukaryotic cells. Although more than 200 N-glycogenes contributing to N-glycan biosynthesis have been identified and characterized, the information on insect N-glycosylation is still limited. Here, focusing on insect N-glycosylation, we characterized Bombyx mori N-acetylgalactosaminyltransferase (BmGalNAcT) participating in complex N-glycan biosynthesis in mammals. BmGalNAcT localized at the Golgi and was ubiquitously expressed in every organ and in the developmental stage of the middle silk gland of fifth instar larvae. Analysis of recombinant BmGalNAcT expressed in Sf9 cells showed that BmGalNAcT transferred GalNAc to non-reducing terminals of GlcNAcβ1,2-R with β1,4-linkage. In addition, BmGalNAcT mediated transfer of galactose and N-acetylglucosamine residues but not transfer of either glucose or glucuronic acid from the UDP-sugar donor substrate to the N-glycan. Despite this tri-functional sugar transfer activity, however, most of the endogenous glycoproteins of insect cells were present without GalNAc, Gal, or GlcNAc residues at the non-reducing terminal of β1,2-GlcNAc residue(s). Moreover, overexpression of BmGalNAcT in insect cells had no effect on N-acetylgalactosaminylation, galactosylation, or N-acetylglucosaminylation of the major N-glycan during biosynthesis. These results suggested that B. mori has a novel multifunctional glycosyltransferase, but the N-glycosylation is highly and strictly regulated by the endogenous N-glycosylation machineries.

scholarly journals The Donor Substrate Specificity of the Human β1,3-Glucuronosyltransferase I toward UDP-Glucuronic Acid Is Determined by Two Crucial Histidine and Arginine Residues

2002 ◽  
Vol 277 (28) ◽  
pp. 25439-25445 ◽  
Author(s):  
Mohamed Ouzzine ◽  
Sandrine Gulberti ◽  
Nicolas Levoin ◽  
Patrick Netter ◽  
Jacques Magdalou ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Glucuronic Acid ◽  
Donor Substrate ◽  
Arginine Residues

Role of Bm30kc6 gene in cell apoptosis and the silk gland degradation signaling pathway in Bombyx mori L.

2020 ◽  
Vol 105 (3) ◽  
Author(s):  
Ying Xiao ◽  
Lei‐lei Li ◽  
Asma Bibi ◽  
Ning Zhang ◽  
Ting Chen ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Signaling Pathway ◽  
Cell Apoptosis ◽  
Silk Gland ◽  
Bombyx Mori L

Sage controls silk gland development by regulating Dfd in Bombyx mori

2021 ◽  
pp. 103568
Author(s):  
Sihan Hou ◽  
Cuicui Tao ◽  
Hongguo Yang ◽  
Tingcai Cheng ◽  
Chun Liu
Keyword(s):  
Bombyx Mori ◽  
Silk Gland ◽  
Gland Development

scholarly journals Vitellogenin receptor selectively endocytoses female-specific and highly-expressed hemolymph proteins in the silkworm, Bombyx mori

2017 ◽  
Vol 95 (4) ◽  
pp. 510-516 ◽  
Author(s):  
Chaoshan Han ◽  
Enxiang Chen ◽  
Guanwang Shen ◽  
Zhixin Peng ◽  
Yinying Xu ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Bombyx Mori ◽  
Sf9 Cells ◽  
Tissue Localization ◽  
Vitellogenin Receptor ◽  
Hemolymph Proteins ◽  
Female Specific ◽  
Oocyte Membrane ◽  
Silkworm Bombyx Mori

VgR, a member of the LDLR family, functions to transport vitellogenin into the ovaries to protome ovarian growth and embryonic development. In insects, the only widely accepted ligand of VgR is Vg. Recently, BmVgR has been shown to interact with BmSP1 in vitro. Therefore, in this study, we evaluated whether BmVgR could transport BmSP1 into certain cells. Although BmVgR could combine with BmVg and BmSP1, BmVgR did not affect the amount of BmSP1 taken up by Sf9 cells. Parallel immunofluorescence showed that most BmVg and BmVgR were localized in the inner oocyte membrane, showing tissue localization similar to that of BmVg labeled with pHrodo Red absorbed by the ovaries on day 2 of pupation. Although BmSP1 showed localization similar to BmVgR during the same phase, little BmSP1 was present in the ovary. Additionally, BmSP1 did not exist in ovaries when the ovaries contained BmVgR on day 5 of pupation, suggesting that BmSP1 in the ovaries was not endocytosed by BmVgR. In summary, BmVgR could facilitate uptake of BmVg by developing oocytes, but did not modulate in the transport of BmSP1.

scholarly journals Comparison of Silks from Pseudoips prasinana and Bombyx mori Shows Molecular Convergence in Fibroin Heavy Chains but Large Differences in Other Silk Components

2021 ◽  
Vol 22 (15) ◽  
pp. 8246
Author(s):  
Michal Rindos ◽  
Lucie Kucerova ◽  
Lenka Rouhova ◽  
Hana Sehadova ◽  
Michal Sery ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Silk Gland ◽  
Amino Acid Sequences ◽  
Phylogenetic Distance ◽  
Rna Seq ◽  
Chain Molecules ◽  
Heavy Chains ◽  
Lepidopteran Larvae ◽  
Crystalline Domains

Many lepidopteran larvae produce silk feeding shelters and cocoons to protect themselves and the developing pupa. As caterpillars evolved, the quality of the silk, shape of the cocoon, and techniques in forming and leaving the cocoon underwent a number of changes. The silk of Pseudoips prasinana has previously been studied using X-ray analysis and classified in the same category as that of Bombyx mori, suggesting that silks of both species have similar properties despite their considerable phylogenetic distance. In the present study, we examined P. prasinana silk using ‘omics’ technology, including silk gland RNA sequencing (RNA-seq) and a mass spectrometry-based proteomic analysis of cocoon proteins. We found that although the central repetitive amino acid sequences encoding crystalline domains of fibroin heavy chain molecules are almost identical in both species, the resulting fibers exhibit quite different mechanical properties. Our results suggest that these differences are most probably due to the higher content of fibrohexamerin and fibrohexamerin-like molecules in P. prasinana silk. Furthermore, we show that whilst P. prasinana cocoons are predominantly made of silk similar to that of other Lepidoptera, they also contain a second, minor silk type, which is present only at the escape valve.

scholarly journals CRISPR-Mediated Endogenous Activation of Fibroin Heavy Chain Gene Triggers Cellular Stress Responses in Bombyx mori Embryonic Cells

Insects ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 552
Author(s):  
Wenbo Hu ◽  
Xiaogang Wang ◽  
Sanyuan Ma ◽  
Zhangchuan Peng ◽  
Yang Cao ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Heavy Chain ◽  
Stress Responses ◽  
Cellular Stress ◽  
Silk Gland ◽  
Cellular Stress Response ◽  
Molar Ratio ◽  
Chain Gene ◽  
Gland Cells ◽  
Cellular Stress Responses

The silkworm Bombyx mori is an economically important insect, as it is the main producer of silk. Fibroin heavy chain (FibH) gene, encoding the core component of silk protein, is specifically and highly expressed in silk gland cells but not in the other cells. Although the silkworm FibH gene has been well studied in transcriptional regulation, its biological functions in the development of silk gland cells remain elusive. In this study, we constructed a CRISPRa system to activate the endogenous transcription of FibH in Bombyx mori embryonic (BmE) cells, and the mRNA expression of FibH was successfully activated. In addition, we found that FibH expression was increased to a maximum at 60 h after transient transfection of sgRNA/dCas9-VPR at a molar ratio of 9:1. The qRT-PCR analysis showed that the expression levels of cellular stress response-related genes were significantly up-regulated along with activated FibH gene. Moreover, the lyso-tracker red and monodansylcadaverine (MDC) staining assays revealed an apparent appearance of autophagy in FibH-activated BmE cells. Therefore, we conclude that the activation of FibH gene leads to up-regulation of cellular stress responses-related genes in BmE cells, which is essential for understanding silk gland development and the fibroin secretion process in B. mori.

Changes in H+-translocating vacuolar-type ATPase in the anterior silk gland cell of Bombyx mori during metamorphosis.

1998 ◽  
Vol 201 (4) ◽  
pp. 479-486
Author(s):  
M Azuma ◽  
Y Ohta
Keyword(s):  
Bombyx Mori ◽  
Gland Cell ◽  
Specific Inhibitor ◽  
Instar Larva ◽  
Silk Gland ◽  
Ultrastructural Changes ◽  
Immunocytochemical Staining ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Silkworm Larvae

A proton-translocating vacuolar-type ATPase (V-ATPase) was identified and characterized in the anterior silk gland of Bombyx mori. By incubating the intact tissue with the fluorescent dye Acridine Orange, the acidified compartment was detected at the apical pole of the epithelial cells. This was observed throughout the feeding period of the fifth-instar larva until the onset of spinning. Acidification was prevented completely and reversibly by 0.8 micromol l-1 bafilomycin A1, a specific inhibitor of V-ATPase. The presence of V-ATPase in a microsomal fraction was verified by immunoblots using an antiserum to the V-ATPase holoenzyme from Manduca sexta midgut. The antiserum localized the V-ATPase to the apical plasma membrane of the anterior silk gland cells, suggesting that the enzyme is functionally active in pumping protons out of the cell towards the glandular lumen of feeding silkworm larvae. In spinning larvae, the acidification produced by the V-ATPase appears to cease, because acidic compartments were seen rarely and only in the periphery of basal cytoplasm, and because immunocytochemical staining for the V-ATPase was greatly reduced at the apical surface. The metamorphic changes in relation to the occurrence of V-ATPase corresponded well with the ultrastructural changes in the anterior silk gland cell of Bombyx mori larvae.

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