Qi ZT et al. (2011), Cloning and expression analysis of a long type ...

XB-ART-43791

Dongwuxue Yanjiu 2011 Aug 01;324:371-8. doi: 10.3724/SP.J.1141.2011.04371.

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Cloning and expression analysis of a long type peptidoglycan recognition protein (PGRP-L) from Xenopus tropicalis.

Qi ZT , Zhang QH , Wang ZS , Wang AM , Huang B , Chang MX , Nie P .

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Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors (PRRs) of the immune system, which bind and hydrolyze bacterial peptidoglycan. Here, a long type PGRP (PGRP-L) was first cloned in the lower vertebrate species Xenopus tropicalis (Xt). The XtPGRP-L possessed a conserved genomic structure with five exons and four introns. The alignment and phylogenetic analysis indicated that XtPGRP-L might be a type of amidase-like PGRP. The 3-D model showed that XtPGRP-L possessed a conserved structure compared with the Drosophila PGRP-Lb. During embryonic development, XtPGRP-L was not expressed until the 72 h tadpole stage. In adult tissues, it was strongly expressed in the liver, lung, intestine, and stomach. Furthermore, after LPS stimulation, the expression of XtPGRP-L was up-regulated significantly in the liver, intestine and spleen, indicating that XtPGRP-L may play an important role in the innate immunity of Xenopus tropicalis.

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Species referenced: Xenopus tropicalis
Genes referenced: pglyrp2

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	Fig. 1 Comparing the XtPGRP-L protein and gene structure with other vertebrates’ long type PGRPs Putative PGRP domains, signal peptides (SP) and transmembrane domains (TM) are indicated. Hm: Homo sapiens; Mm: Mus musculus; Xe: Xenopus tropicalis; Dr: Danio rerio.
	Fig. 2 Multiple alignment of XtPGRP-L with other PGRPs Amino acid sequences were extracted from GenBank, and the alignment was produced with CLUSTL W. The three levels of shading indicate 100%, 80%, or 60% similar amino acids decorated with the GeneDoc program. The amino acids needed for T7 amidase are marked with dots. The GenBank accession no. of all sequences used are listed in Tab. 2.
	Fig. 3 The predicted secondary structure and homology modeling of XtPGRP-L a) 2-D structure of XtPGRP-L. The conserved structures for all PGRPs are indicated as α1-3 and α3-7. The α* is the specific region of XtPGRP-L. b) The 3-D structure of XtPGRP-L. The cysteines for the S-S bridge were labelled according to the Dm PGRP-Lb as cys46, cys52.
	Fig. 4 An unrooted phylogenetic tree constructed by the neighbor joining from amino acid sequences of PGRPs together with the XtPGRP-L gene The number indicates the bootstrap confidence values obtained for each node after 1000 replications. The GenBank accession no. of all sequences used are listed in Tab. 2.
	Fig. 5 Expression pattern of the XtPGRP-L gene in developing embryos, tissues of normal Xenopus and LPS stimulated Xenopus (a) Expression of XtPGRP-L in developing embryos. (b) Tissue distribution of XtPGRP-L in normal Xenopus. β-actin was used as a control for the amount and quality of cDNA. Lines 1 to 7 represent spleen, liver, kidney, intestine, gall bladder, fat body, and stomach, respectively. (c) Western blot analysis of the XtPGRP-L in normal Xenopus tissues. (d) Quantitative PCR analysis of the XtPGRP-L expression in normal Xenopus tissues. (e) Quantitative PCR analysis of the XtPGRP-L expression change after LPS stimulation. Asterisks indicate that the copy number is significantly higher than that of the corresponding control sample (*P < 0.05).