Genes encoding proteins with peritrophin A-type chitin-binding domains in Tribolium castaneum are grouped into three distinct families based on phylogeny, expression and function

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Abstract

This study is focused on the characterization and expression of genes in the red flour beetle, Tribolium castaneum, encoding proteins that possess one or more six-cysteine-containing chitin-binding domains related to the peritrophin A domain (ChtBD2). An exhaustive bioinformatics search of the genome of T. castaneum queried with ChtBD2 sequences yielded 13 previously characterized chitin metabolic enzymes and 29 additional proteins with signal peptides as well as one to 14 ChtBD2s. Using phylogenetic analyses, these additional 29 proteins were classified into three large families. The first family includes 11 proteins closely related to the peritrophins, each containing one to 14 ChtBD2s. These are midgut-specific and are expressed only during feeding stages. We propose the name “Peritrophic Matrix Proteins” (PMP) for this family. The second family contains eight proteins encoded by seven genes (one gene codes for 2 splice variants), which are closely related to gasp/obstructor-like proteins that contain 3 ChtBD2s each. The third family has ten proteins that are of diverse sizes and sequences with only one ChtBD2 each. The genes of the second and third families are expressed in non-midgut tissues throughout all stages of development. We propose the names “Cuticular Proteins Analogous to Peritophins 3” (CPAP3) for the second family that has three ChtBD2s and “Cuticular Proteins Analogous to Peritophins 1 (CPAP1) for the third family that has 1 ChtBD2. Even though proteins of both CPAP1 and CPAP3 families have the “peritrophin A” domain, they are expressed only in cuticle-forming tissues. We determined the exon–intron organization of the genes, encoding these 29 proteins as well as the domain organization of the encoded proteins with ChtBD2s. All 29 proteins have predicted cleavable signal peptides and ChtBD2s, suggesting that they interact with chitin in extracellular locations. Comparison of ChtBD2s-containing proteins in different insect species belonging to different orders suggests that ChtBD2s are ancient protein domains whose affinity for chitin in extracellular matrices has been exploited many times for a range of biological functions. The differences in the expression profiles of PMPs and CPAPs indicate that even though they share the peritrophin A motif for chitin binding, these three families of proteins have quite distinct biological functions.

Introduction

Chitin, an extracellular matrix polysaccharide composed of β(1  4) linked N-acetylglucosamine residues, is a major component of the insect exoskeleton, tracheae and midgut peritrophic matrix (PM). In all of these structures, chitin is associated with an assortment of proteins that influence the physical, chemical and biological properties of the extracellular structure. Although the exact chemical nature of the association of the chitinous matrix with proteins is not well understood, progress is being made in cataloging the proteins found in the cuticle and PM. Two main classes of chitin-binding motifs have been identified in insect proteins. The first class contains a sequence consisting of six cysteines that probably form three disulfide bridges. This sequence motif is referred to as the “peritrophin A domain” and is found in numerous proteins extracted from insect PMs. This motif belongs to the CBM14 family of carbohydrate-binding domains (pfam 01607; Elvin et al., 1996) also known as the type 2 chitin-binding domain (ChtBD2 = SMART 00494). The second general class of chitin-binding sequence motifs found in insect cuticular proteins is the Rebers and Riddiford Consensus sequence (R&R Consensus; PDGDYNY+YETSNGIADQETGD+KSQGETRDG++AVDVV+GSYSYVDPDGTTRTVTYTADDENGFQPVGAHLP; pfam00379; Rebers and Riddiford, 1988, Andersen, 2010, Karouzou et al., 2007, Willis, 2010), which is devoid of cysteine residues. Many of these proteins are likely to be cross-linked with one another via quinones derived from N-β-alanyldopamine and N-acetyldopamine (Kramer et al., 2001, Andersen, 2010). Some of these R&R proteins were identified in insect cuticle extracts by proteomic analyses (He et al., 2007).

Evidence for the involvement of the ChtBD2 motif as well as the R&R Consensus in chitin-binding comes from experiments showing a gain in affinity for chitin following attachment of these sequences to a protein that normally does not bind to chitin (Arakane et al., 2003, Rebers and Willis, 2001). Models have been proposed to explain some of the interactions between the N-acetylglucosamines and the amino acid residues lining the putative binding pockets (Kramer and Muthukrishnan, 2005, Hamodrakas et al., 2002, Iconomidou et al., 2005), but the molecular details involved in chitooligosaccharide binding are still unknown for either type of CBD.

Proteins with CBDs that have six cysteine residues with a characteristic spacing between them were initially extracted from the insect PM using strong denaturing agents and were appropriately denoted as “peritrophins” (Tellam et al. 1999; Shen and Jacobs-Lorena, 1999). Later on, Barry et al. (1999) and Behr and Hoch (2005) identified cDNAs/genes encoding proteins with this peritrophin A-type chitin-binding motif, ChtBD2, in cuticle-forming tissues from Drosophila melanogaster. These proteins have three ChtBD2s separated by spacers of characteristic lengths. Gaines et al. (2003) characterized five cDNA clones from RNA expressed in the hindgut and Malpighian tubules of the cat flea, Ctenocephalides felis, which encode proteins with one to four peritrophin A domains. More recently, Nisole et al. (in press) have cloned a cDNA from Choristoneura fumiferana, which encodes a protein homologous to these proteins. This gene was highly expressed in the epidermis and the purified recombinant protein bound to chitin. Thus, it is clear that ChtBD2 is present in proteins that are found in locations other than the PM. However, the group of proteins isolated from the PM after extraction with strong denaturing agents, collectively called “peritrophins,” exhibit a wider variation in the number of ChtBD2s and, consequently, in molecular masses. Several other proteins with a large number of ChtBD2 repeats have been predicted from genome sequences of insects, even though they have not been extracted directly from PM or gut tissue. The number of ChtBD2s in “peritrophins” can range from one to 19 (reviewed in Tellam et al., 1999, Dinglasan et al., 2009, Venancio et al., 2008, Hegedus et al., 2009). Many of these are also interspersed with serine and threonine-rich mucin-like domains that are likely to be glycosylated (Toprak et al. 2009). These studies have revealed that the group of ChtBD2-containing proteins is much larger than previously known. However, a bioinformatics study cataloging all proteins with one or more ChtBD2s (regardless of their location) encoded by a single genome has not been undertaken so far. In this paper, we have used an exhaustive bioinformatics search to identify in the red flour beetle, Tribolium castaneum, all of the genes that encode proteins containing one or more ChtBD2s, which have been associated with the ability to bind to chitin. Our studies have revealed that the T. castaneum proteome contains a large assortment of proteins with ChtBD2s in addition to the PM-associated peritrophins and enzymes of chitin metabolism such as chitinases and chitin deacetylases, some of which have ChtBD2s. A vast majority of them have signal peptide sequences at the N-terminus, a characteristic consistent with their roles in binding to chitin in extracellular matrices. We propose a new nomenclature for three gene families that encode non-enzymatic proteins with one or more ChtBD2s, the first being Peritrophic Matrix Proteins (PMPs) for proteins expressed in the midgut, and the second and third as “Cuticular Proteins Analogous to Peritophins 1 and 3” (CPAP1 and CPAP3) for proteins expressed in cuticle-forming tissues with one and three ChtBD2 domains respectively.

Section snippets

Insect cultures

The GA1 strain of T. castaneum was used in all experiments. Insects were reared at 30 °C in wheat flour containing 5% brewer's yeast under standard conditions as described previously (Beeman and Stuart, 1990). The GA1 strain used in our study is a highly inbred robust strain from which the GA2 strain was obtained and used for DNA sequencing.

Identification and cloning of the genes encoding proteins with ChtBD2s

Sequence comparisons of previously characterized peritrophins from several insects including T. castaneum indicated substantial variations in the spacing

Bioinformatics search of T. castaneum genome databases

The initial search of the Tribolium genome identified 29 proteins with ChtBD2s. The domains from these proteins were subsequently used in a second search to identify additional proteins with ChtBD2s. This process was repeated until no additional protein sequences with ChtBD2s could be identified. In the end, we identified a total of 49 putative T. castaneum genes capable of encoding 50 proteins (one gene codes for two proteins as a result of alternative splicing) with one or more ChtBD2s. Of

Discussion

The goal of this research was to carry out a comprehensive search for T. castaneum proteins that contain one or more ChtBD2s with the expectation that these proteins, which are predicted to interact with chitin, will have a role in assembly and/or turnover of chitin-containing structures. This domain was found originally in several proteins known as peritrophins that were extracted from the PM and shown to bind to chitin tightly (Tellam et al., 1999, Wang et al., 2004). It has been proposed

Acknowledgments

We thank Kathy Leonard for beetle husbandry and Dr. Qingsong Zhu for some of the preliminary work on CPAP3. We thank Neal Dittmer for his critical review of this paper and for valuable suggestions. This project was supported by NSF grants IBN-0316963 and IOS-615818. This is contribution no. 10-115-J from the Kansas Agricultural Experiment Station. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply

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