Molecular cloning, gene expression analysis, and in silico characterization of UDP‐N‐acetylglucosamine pyrophosphorylase from Bombyx mori

Abstract

The present study was aimed to explore the molecular and structural features of UDP‐N‐acetylglucosamine pyrophosphorylase of Bombyx mori (BmUAP), an essential enzyme for chitin synthesis in insects. The BmUAP cDNA sequence was cloned and expression profiles were monitored during the molting and feeding stages of silkworm larvae. The effect of 20‐hydroxyecdysone (20E) on BmUAP expression, and on silkworm molting was studied, which revealed that 20E regulates its expression. Multiple sequence alignment of various pyrophosphorylases revealed that the residues N223, G290, N327, and K407 of human UAP (PDB ID: 1JV1) were found to be highly conserved in BmUAP and all other eukaryotic UAPs considered for the study. Phylogenetic analysis inferred that the UAPs possess discrete variations in primary structure among different insect Orders while sharing good identity between species of the Order. The structure of BmUAP was predicted and its interactions with uridine triphosphate, N‐acetylglucosamine‐1‐phosphate, and UDP‐N‐acetylglucosamine were analyzed. Virtual screening with a library of natural compounds resulted in five potential hits with good binding affinities. On further analysis, these five hits were found to be mimicking substrate and product, in inducing conformational changes in the active site. This work provides crucial information on molecular interactions and structural dynamics of insect UAPs.

Silencing uridine diphosphate N‐acetylglucosamine pyrophosphorylase gene impairs larval development in Henosepilachna vigintioctopunctata

“…Third, our data revealed that trachea had the highest HvUAP level, followed by foregut, midgut, hindgut and epidermis in H. vigintioctopunctata (Figure 1). Similar expression profiles of UAP have been observed in other insects 10 . In L. migratoria , for example, both LmUAP1 and LmUAP2 are widely expressed in the epidermis, foregut, midgut, hindgut, gastric caeca, Malpighian tubules, wing, trachea, fat body and muscle 28 .…”

“…Similar expression profiles of UAP have been observed in other insects. 10 In L. migratoria, for example, both LmUAP1 and LmUAP2 are widely expressed in the epidermis, foregut, midgut, hindgut, gastric caeca, Malpighian tubules, wing, trachea, fat body and muscle. 28 Correspondingly, TcUAP1 transcripts are widely distributed in larval midgut, hindgut, carcass (the entire body exclusive of gut), fat body and integument in T. castaneum.…”

Molecular Characterization of UDP-N-Acetylglucosamine Pyrophosphorylase and Its Role in the Growth and Development of the White-Backed Planthopper Sogatella furcifera (Hemiptera: Delphacidae)

“…Furthermore, SfUAP expression significantly increased after each nymph molting, decreased during the inter-molting phase, and increased again before the next molt, which may be associated with nymph growth. This phenomenon was observed in L. migratoria and B. mori , in which UAP expression was periodically repeated at each molting cycle [ 25 , 34 ]. Additionally, we noted that the expression pattern of SfUAP was similar to that of chitin synthase, which is responsible for chitin formation in T. castaneum , Ostrinia furnacalis , N. lugens , B. mori , and S. furcifera [ 40 , 41 , 44 , 45 , 46 ].…”

“…UAP (EC 2.7.7.23), a vital regulatory enzyme in the insect chitin biosynthesis pathway, specifically catalyzes the reaction of N -acetylglucosamine-1-phosphate with uridine triphosphate to yield UDP- N -acetylglucosamine (UDP-GlcNAc), which is an important substrate for the formation of chitin [ 24 , 26 ]. So far, UAPs have been isolated and characterized from several insect species, including Locusta migratoria [ 25 ], Bactrocera dorsalis [ 27 ], Tribolium castaneum [ 28 ], Spodoptera exigua [ 29 ], Aedes aegypti [ 30 ], Cnaphalocrocis medinalis [ 31 , 32 ], Leptinotarsa decemlineata [ 33 ], Bombyx mori [ 34 ], and Henosepilachna vigintioctopunctata [ 35 ]. Interestingly, most insects have been known to only possess a single UAP gene, except T. castaneum , L. migratoria , and L. decemlineata , all of which have two UAP genes ( UAP1 and UAP2 ).…”

Global Analysis of UDP Glucose Pyrophosphorylase (UDPGP) Gene Family in Plants: Conserved Evolution Involved in Cell Death

“…Some studies on UAP were reported to illustrate its function in microorganisms, animals, and plants. UAP was reported in both prokaryotes and eukaryotes, while no homologous sequences were identified between them ( Palaka et al, 2019 ). In fungi, a singular UDP-GlcNAc pyrophosphorylase gene was reported in yeast ( Saccharomyces cerevisiae ), and loss-of-function mutant ( uap1 Δ) exhibited aberrant morphology including swelled or lysed ( Mio et al, 1998 ).…”