Reproductive toxicology. Ethylene glycol.

Background: Chitinases (EC.3.2.1.14) hydrolyze the β-1,4-linkages in chitin, an abundant Nacetyl-β-D-glucosamine polysaccharide that is a structural component of protective biological matrices such as insect exoskeletons and fungal cell walls. The glycoside hydrolase 18 (GH18) family of chitinases is an ancient gene family widely expressed in archea, prokaryotes and eukaryotes. Mammals are not known to synthesize chitin or metabolize it as a nutrient, yet the human genome encodes eight GH18 family members. Some GH18 proteins lack an essential catalytic glutamic acid and are likely to act as lectins rather than as enzymes. This study used comparative genomic analysis to address the evolutionary history of the GH18 multiprotein family, from early eukaryotes to mammals, in an effort to understand the forces that shaped the human genome content of chitinase related proteins. Results: Gene duplication and loss according to a birth-and-death model of evolution is a feature of the evolutionary history of the GH18 family. The current human family likely originated from ancient genes present at the time of the bilaterian expansion (approx. 550 mya). The family expanded in the chitinous protostomes C. elegans and D. melanogaster, declined in early deuterostomes as chitin synthesis disappeared, and expanded again in late deuterostomes with a significant increase in gene number after the avian/mammalian split. Conclusion: This comprehensive genomic study of animal GH18 proteins reveals three major phylogenetic groups in the family: chitobiases, chitinases/chitolectins, and stabilin-1 interacting chitolectins. Only the chitinase/chitolectin group is associated with expansion in late deuterostomes. Finding that the human GH18 gene family is closely linked to the human major histocompatibility complex paralogon on chromosome 1, together with the recent association of GH18 chitinase activity with Th2 cell inflammation, suggests that its late expansion could be related to an emerging interface of innate and adaptive immunity during early vertebrate history. Background The recent increase in genome projects has provided DNA sequence data useful for understanding the evolutionary dynamics that resulted in the conservation of ancient proteins as modern protein families. One such family of proteins is the glycoside hydrolase family 18 chitinases (GH18), widely expressed in archea, prokaryotes and eukaryotes. Definition of this family is based on amino acid sequence similarity [1-3]. In eukaryotes the proteins are mainly expressed by fungi, arthropods and nematodes Published: 26 June 2007 BMC Evolutionary Biology 2007, 7:96 doi:10.1186/1471-2148-7-96 Received: 6 November 2006 Accepted: 26 June 2007 This article is available from: http://www.biomedcentral.com/1471-2148/7/96 © 2007 Funkhouser and Aronson; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Page 1 of 16 (page number not for citation purposes) BMC Evolutionary Biology 2007, 7:96 http://www.biomedcentral.com/1471-2148/7/96 (for review see [4]), but they are also expressed in mammals, with members recently shown to be involved in important physiological processes that include T-cell mediated inflammation and allergy [5,6]. The chitinases hydrolyze chitin, an abundantly produced homopolymer of N-acetyl-β-D-glucosamine that provides architectural reinforcement of biological structures such as insect exoskeletons, fungal cell walls, nematode egg shells, and other biological matrices involved in protection and self defense. Chitin has not been detected in higher plants and vertebrates, where cellulose and hyaluronan, respectively, may replace certain chitin functions [7,8]. In chitinous species, the chitinases along with chitin synthetases are essential for remodeling chitin-containing structures during growth and development. Certain bacterial genera and plants, which do not produce chitin, use chitinases, respectively, for the conversion of insoluble chitin into metabolizable nutrient sources and for defense from chitinous pathogens. Mammals are not known to synthesize chitin or to metabolize it as a nutrient, yet the human genome encodes eight well-documented genes for proteins now classified as glycoside hydrolase family18 members. Members of this family are known to adopt the TIM (triosephosphate isomerase) fold consisting of a strongly conserved (β/α)8barrel structure [1-3]. Often, separate chitin-binding domains (CBM14) [2,3] are present in the carboxyl terminal region of the proteins (additional file 1: GH18 family domain structure). The protein family includes chitinases as well as homologous proteins termed chitolectins. The latter lack the key active-site glutamate residue that donates a proton required for hydrolytic enzyme activity, but retain highly conserved residues involved in oligosaccharide binding and overall three-dimensional structure. Traditionally, chitinases are classified in two glycoside hydrolase families, GH18 and GH19, with different structures and catalytic mechanisms [9]. Family GH18 includes the chitinases from viruses, bacteria, fungi and animals as well as classes III and V from plants [10]. The GH19 chitinases are identified mostly in plants (classes I, II and IV), nematodes, and some bacteria [11]. Recent data indicate chitinase activity is also present in protein families GH48 and GH20 [12,13]. N-acetyl-β-D-glucosaminidases such as those in family GH20 also can participate in chitin degradation by hydrolyzing GlcNAc from the non-reducing end of chito-oligosaccharides [14]. Three of the GH18 proteins encoded by the human genome have demonstrated enzymatic activity. They are: di-N-acetylchitobiase, active in the lysosomal degradation of asparagine-linked glycoproteins [15]; chitotriosidase, a vesicular and secreted protein produced by activated macrophages and highly elevated in serum from patients with Gaucher disease [16]; and acidic mammalian chitinase (AMCase), expressed mostly in the gastrointestinal tract and lung [17], and recently shown to be induced by Th2helper cells in an aeroallergen model of asthma [5]. A gene similar to the AMCase gene encodes a fourth hypothetical protein with a truncated GH18 domain. Three human genes (OVGP1, CHI3L1 and CHI3L2) encode chitolectins likely to be involved in tissue remodeling during inflammation and/or development. The OVGP1 gene encodes a large glycoprotein secreted by oviduct epithelial cells in response to estrogen [18,19]. The glycoprotein includes an extended C-terminal repeat region similar to sequences found in mucins. A number of cell types, including macrophages, articular chrondrocytes and synovial cells, secrete proteins encoded by the CHI3L1 and CHI3L2 genes under inflammatory conditions [20-22]. A recently identified CHID1 gene encodes a lysosomal GH18 chitolectin that interacts with stabilin-1, an endocytic/sorting receptor of macrophages that serves as a marker of alternative activation [23]. The function of the CHID1 protein is not yet established, and the sequence similarity to other GH18 proteins is low. Although the biological functions of most human GH18 proteins are not yet delineated, the available protein characterizations indicate that a process of gene duplication and diversification has resulted in multiple functions that are not related to nutrient utilization or growth-related turnover of chitinous structures. Instead, recent data pertaining to the mammalian proteins point to their prominent roles in defense against fungal or other pathogens and in inflammation and remodeling processes [6]. In this communication, we addressed the evolutionary history of the GH18 multiprotein family from early eukaryotes to mammals in an effort to understand the forces that shaped the human genome content of chitinase-related proteins. The present study indicates that over evolutionary time the GH18 family evolved by decline and expansion according to selective forces associated with speciation. An expansion of chitinase genes occurred as chitinous species appeared early in metazoan evolution, a decline followed as chitin disappeared as an important structural and protective feature of early deuterostomes, and a second expansion began in non-chitinous vertebrates where chitinases and chitolectins may have evolved to assume increasingly important roles associated with pathogen recognition, processing and antigen presentation. Results Human GH18 Family Genes and Their Relationship to Other Mammalian GH18 Genes Seven of the eight human GH18 family members are located on chromosome 1. The chitobiase gene is located at 1p22, 26 Mb upstream of the regions containing six other chitinase/chitolectin genes, which cluster into two Page 2 of 16 (page number not for citation purposes) BMC Evolutionary Biology 2007, 7:96 http://www.biomedcentral.com/1471-2148/7/96 groups, at 1p13 and at 1q32 (Table 1): Genes CHI3L2, CHIA, RP11-165H20.1 and OVGP1 cluster at 1p13; and CHIT1 and CHI3L1 at 1q32. Only the recently annotated and weakly homologous CHID1 gene is not located on chromosome 1, but at chromosome 11p15.5. A similar but unique pattern of homologues is present in the mouse genome, which includes ten genes encoding GH18 family members. The genes are located on mouse chromosomes 1, 3 and 7. Genes Ctbs, Chia, Chi3l3, Chi3l4, Ovgp1, Bclp2 and BC051070 are located on chromosome 3, and Chit1 and Chi3l1 are on chromosome 1. The mouse CHID1 homolog, designated 3110023E09Rik [Entrez Gene ID 68038], is located at chromosome 7F5. The rat genome is not complete, but homologous rat genes are located on chromosomes 2, 13 and 18. There is conservation of synteny in the chromosomes of these three mammals that contain GH18 family members. From the genome sequences currently available, it is evident based on protein sequence similarities that the mammalian genomes include orthologous clades of GH18 family members, and that the chitinase/chitolectin clade (Figure 1) includes orthologous groups of paralogous family members. Construction of a phylogenetic tree from alignment of GH18 domains from 36 mammalian proteins (additional file 2: Mammalian Sequences) illustrates these relationships. Figure 1 shows the tree, constructed using the minimum evolution method. The lysosomal chitobiases and stabilin-1 interacting chitolectins form outlying clades of orthologous proteins distinct from the true chitinases and chitolectins, which form four separate subgroups. Subgroup I represents the acidic chitinases and related chitolectins; subgroup II, the chitotriosidase enzymes; subgroup III, the non-enzymatic proteins associated with injury, repair and remodeling; and subgroup IV, the oviduct glycoproteins and related sequences. Each of the groups is not represented in all the genomes. This may be explained by genome projects that are not complete; or gene duplication or gene loss may have occurred relatively late in vertebrate evolution. Among GH18 enzymes, chitobiase is biochemically unique as the only member that splits off a monosaccharide from the reducing end of chito-oligosaccharides [15]. Chitinase/Chitolectin Genes in Early Eukaryotes (D. discoideum and H. echinata) To assess the family GH18 genes in early eukaryotes and their relationship to the human genes, we examined the genome of the social amoeba Dictyostelium discoideum. D. discoideum diverged from the animal-fungal lineage after the plant-animal split, and has retained a significant amount of the diversity of the ancestral eukaryotic genome [24]. Thus, chitinases present in the D. discoideum genome should provide evolutionary insight into the ancestral metazoan GH18 proteins present before an apparent expansion of chitinases associated with the biological importance of chitin in protostomes (discussed in the next section). A search of the D. discoideum genome database at NCBI (Build 1.1;November 22, 2005) revealed six genes encoding chitinase-related proteins. Five of the genes cluster on two chromosomes: a 2.6 Mbp region of chromosome 2 encodes Entrez genes 3393954, 3395463, and 3394351; and a 3.5 Kb region of chromosome 5 encodes Entrez genes 3388787 and 3388788. A sixth gene (Entrez Gene 3387295) also located on chromosome 5 encodes a hypothetical protein [GenBank:XP_635730] that aligned with the human stabilin-1 interacting protein (Expect = 2e-48). Phylogenetic analysis (Figure 2) indicated that the three chromosome 2 genes are co-orthologs of the human chitobiase. Two chromosome 5 genes are more distant homologs, distinct from the human chitinases/chitolectins. The third chromosome 5 gene is a human CHID1 ortholog. If the eukaryotic ancestral genome included orthologs of the mammalian chitinase/chitolectin group, and the presence of chitinases in plants [11] indicates it did, they likely have been lost in the D. discoideum lineage. As the cells of Dictostelium are professional phagocytes that consume Table 1: Human Genes Encoding GH18 Family Members Entrez Gene ID Gene (Aliases) Chra Location Protein Accession Description 27159 CHIA 1p13.2 NP_970615 & NP_068569 acidic chitinase (AMCase, eosinophil chemotactic cytokine) 1118 CHIT1 1q32.1 NP_003456 chitinase 1 (chitotriosidase) 1116 CHI3L1 (YKL 40, GP39, TSA1902) 1q32.1 NP_001267 chitinase 3-like 1 (cartilage protein 39) 1117 CHI3L2 (YKL 39) 1p13.3 NP_003991 chitinase 3-like 2 (chondrocyte protein 39) RP11-165H20.1 (LOC149620) 1p13.2 NP_001013643 NCBI Ref Seq: NP_001013643 was permanently suppressed because it is a nonsense mediated mRNA decay (NMC) candidate 5016 OVGP1 1p13.2 NP_002548 oviductal glycoprotein 1; oviductin 1486 CTBS 1p22 NP_004379 chitobiase, di-N-acetyl 66005 CHID1 11p15.5 NP_076436 stabilin-1 interacting chitinase-like protein