The family of amphioxus chitin synthases offers insight into the evolution of chitin formation in chordates

https://doi.org/10.1016/j.ympev.2019.106691Get rights and content

Highlights

  • Amphioxus has the biggest chitin synthase (CS) gene pool in eukaryotes.

  • Amphioxus is the only chordate having both type-I and type-II CSs.

  • Amphioxus is the only metazoan with a greatly expanded type-I CS lineage.

  • Type-I CSs were lost early in urochordates and vertebrates.

  • Type-II CSs was split into two lineages but finally all lost in higher vertebrates.

Abstract

Chitin is a very important and widely-used biopolymer in fungi and lower metazoans, but mysteriously disappears in mammals. Recent studies reveal that at least lower vertebrates have chitin synthases (CS) and use them to synthesize endogenous chitin. Amphioxus, a basal chordate, therefore becomes critical to understand the evolution of CS, as it occupies the transitional position from invertebrates to vertebrates, and is considered as a good proxy to the chordate ancestor.

Here, by exploiting multiple genome assemblies, high-depth RNA-seq data and synteny relations, we identify 11–12 CS genes for each amphioxus species. It represents the largest CS gene pool ever found in eukaryotes so far. As comparison, most metazoans have one or two CSs. Amphioxus is the only chordate that has both the very ancient type-I CS family and the more broadly distributed type-II CS family. Specifically, amphioxus has only one type-II CS but 10–11 type-I CSs, which means that amphioxus is the only metazoan with a greatly expanded type-I CS family. Further analysis suggests that the chordate ancestor have at least one type-II CS and an expanded of type-I CS family. We hypothesize that: these ancient CSs are mostly retained in amphioxus; but the whole type-I CS family was lost in urochordates and vertebrates; the type-II CS was later duplicated into two lineages in vertebrates and followed by stochastic losses, till all type-II CSs were eventually lost in birds and mammals. Finally, our expression profiling and preliminary gene knockout analysis suggest that amphioxus CSs could have highly diverse but mildly overlapping functions in various tissues and organs.

Taken together, these findings not only provide insights into the evolution of chordate CSs, lay a foundation for further functional study of the chordate CSs. After all, it is mysterious that our chordate ancestor needed so many isoenzymes for chitin formation.

Introduction

Chitin is one of the most abundant polymers and the most widespread amino polysaccharides in nature (Kumar, 2000). Chitin is synthesized by a wide range of organisms. In fungi, diatoms and other unicellular eukaryotes, chitin involves in cell wall formation and some other functions (Bowman and Free, 2006, Durkin et al., 2009, Herth et al., 1977, Mulisch and Hausmann, 1989). In Metazoa, chitin is best known and most studied as basic components of body cuticles, exoskeletons and gut peritrophic matrix in arthropods (Hegedus et al., 2009, Rudall and Kenchington, 1973). There are also reports that chitin participates in the formation of molluscan teeth and annelid chaetae, and in the biomineralization in poriferans, cnidarians, brachiopods and molluscs (Bo et al., 2012, Ehrlich, 2010, Luo et al., 2015, Peters, 1972, Peters and Latka, 1986, Picken and Lotmar, 1950).

Chitin is synthesized by the chitin synthase (CS), a member of glycosyltransferase family 2, which features a variable number (up to 17) of transmembrane regions at both ends and a conserved central catalytic domain (Merzendorfer, 2011). Chitin formation includes three steps (Merzendorfer, 2006): the cytoplasmic part of the catalytic domain forms the polymer by adding uridine diphosphate N-acetylglucosamine (UDP-GlcNac) to the growing oligosaccharide; the nascent polymer is then translocated across the membrane and released to the extracellular space; finally, released single polymers spontaneously assemble into crystalline micro-fibrils.

It was once thought that in Metazoa, only non-chordate invertebrates have CSs and synthesize chitin. But recent phylogenomic analyses reveal that there is actually broad taxonomic distribution and extensive diversification of CSs in metazoans, including chordate invertebrates and vertebrates, except birds and mammals (Tang et al., 2015, Zakrzewski et al., 2014). Metazoan CSs can be divided into type-I and type-II clades. The type-I clade appears the most ancient lineage, because it not only contains all CSs from two basal metazoans (poriferans and cnidarians), but also includes all CSs from choanoflagellates, the putative closest unicellular relative of metazoans. Some lophotrochozoan CSs also fall in the clade, suggesting the broad existence of this clade. One lineage-specific feature of type-I CSs is that their C-terminal region often contains one or two sterile alpha motifs (SAM), which is reportedly important for protein interactions (Thanos et al., 1999).

The type-II clade comprises most CSs from lophotrochozoans, and all CSs from ecdysozoans, urochordates and vertebrates (Zakrzewski et al., 2014). Lophotrochozoan type-II CSs could be divided into four ancient groups. Three of them acquired an N-terminal myosin motor domain (MMD) able to interact with actin cytoskeleton (Tsuizaki et al., 2009). Expansion of type-II CSs is frequent in Lophotrochozoans. For example, mollusc Lottia gigantea has ten CSs, the biggest pool of CS genes observed before this study (Zakrzewski et al., 2014). On the other side, ecdysozoans like nematodes and insects generally have two CSs, with differential functions in the cuticle, the peritrophic membrane and the eggshell (Merzendorfer, 2006, Veronico et al., 2001, Zhu et al., 2002). As for chordates, it appears that some bony fishes have two CS genes, whereas urochordates and amphibians have only one CS (Tang et al., 2015). Like the role of insect CSs in the midgut peritrophic matrix, fish and urochordate CSs play a critical part in the formation of chitin-based barrier immunity in the gut (Nakashima et al., 2018).

Current knowledge on chitin synthesis and functions largely comes from fungi and insects (Merzendorfer, 2011). However, considering the wide distribution, extensive diversification and taxon-specific expansion of CSs in metazoans, the regulation and functions of animal chitin synthesis could be much more complex and diverse than current data suggest (Zakrzewski et al., 2014).

The cephalochordate amphioxus could be exploited to gain more understanding of the evolution of CSs from invertebrates to vertebrates. Amphioxus represents the basal lineage among three chordate subphyla (cephalochordates, urochordates and vertebrates), hence occupying a pivotal transitional taxon connecting invertebrates and vertebrates (Delsuc et al., 2006, Huang et al., 2014b, Putnam et al., 2008). An early report discovered multiple CS gene fragments from the draft genome of amphioxus Branchiostoma floridae (Guerriero, 2012). But many questions remain opened due to the complex gene structure of CSs, the limited expression evidence, and the high rates of assembly and prediction errors in the draft genome of B. floridae. In this study, we identify all possible CS genes from the genomes of amphioxus B. floridae and B. belcheri, which diverged ~120 million years ago (Huang et al., 2014b). Then we try to infer the evolutionary history of the amphioxus CS family by leveraging information from genomes, transcriptomes, synteny, expression profiles and preliminary gene knockout analysis. Our findings provide new insights into the functional evolution of chordate CSs.

Section snippets

Identification of CS gene models from two amphioxus species

A typical CS gene encodes a protein with over 1200 amino acid residues and a variable number of transmembrane regions, and usually has complex exon-intron structures and flexible alternative splicing sites. Moreover, the draft genomes of amphioxus are notorious for excess assembly and prediction errors due to high heterozygosity (Huang et al., 2012, Huang et al., 2017). These situations make the prediction of amphioxus CS genes a non-trivial task. Here we first used homology-based methods to

Conclusions

Here we show that amphioxus have 11–12 CS genes, which is the largest CS gene pool in eukaryotes ever found so far. Many of these genes appear to have ancient origins, probably date back to the latest common chordate ancestor in the Cambrian period. Amphioxus is the only chordate, and one of very few metazoans, that have both type-I and type-II CS genes. Like other chordates that have one or two type-II CSs, amphioxus has only one type-II CS. However, amphioxus has a greatly expanded type-I CS

Sequence resources and searching methods

CS sequences for different species were identified by performing homology-based searches in a multi-stage manner. For amphioxus, known representive B. floridae CSs (NCBI Reference Sequence: XP_002592459 and XP_002592461) (Guerriero, 2012) were used as search queries. 24 putative full-length CSs in B. floridae and B. belcheri genomes were identified by performing BLASTP searches against the B. floridae filtered gene models database (version 1 and 2, //genome.jgi.doe.gov/pages/blast-query.jsf%3fdb%3dBrafl1

Acknowledgements

This work was supported by Marine S&T Fund of Shandong Province (2018SDKJ0302-2), NNSF projects (31971107, 31722052 & 31872595), National Key R&D program of China (2018YFD0900503), Guangdong province project (2017B030314021 & 201804010434) and by National Supercomputer Center in Guangzhou and the Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund.

References (44)

  • S.M. Bowman et al.

    The structure and synthesis of the fungal cell wall

    BioEssays

    (2006)
  • F. Delsuc et al.

    Tunicates and not cephalochordates are the closest living relatives of vertebrates

    Nature

    (2006)
  • C.A. Durkin et al.

    Chitin in diatoms and its association with the cell wall

    Eukaryot. Cell

    (2009)
  • H. Ehrlich

    Chitin and collagen as universal and alternative templates in biomineralization

    Int. Geol. Rev.

    (2010)
  • R.D. Finn et al.

    The Pfam protein families database: towards a more sustainable future

    Nucl. Acids Res.

    (2016)
  • E. Gaya et al.

    The adaptive radiation of lichen-forming Teloschistaceae is associated with sunscreening pigments and a bark-to-rock substrate shift

    Proc. Natl. Acad. Sci. USA

    (2015)
  • D. Hegedus et al.

    New insights into peritrophic matrix synthesis, architecture, and function

    Annu. Rev. Entomol.

    (2009)
  • W. Herth et al.

    Chitinous fibrils in the lorica of the flagellate chrysophyte Poteriochromonas stipitata (syn. Ochromonas malhamensis)

    J. Cell Biol.

    (1977)
  • L.Z. Holland

    Amphioxus genomics

    Brief Funct. Genom.

    (2012)
  • G. Huang et al.

    Two apextrin-like proteins mediate extracellular and intracellular bacterial recognition in amphioxus

    Proc. Natl. Acad. Sci. USA

    (2014)
  • S. Huang et al.

    HaploMerger: reconstructing allelic relationships for polymorphic diploid genome assemblies

    Genome Res.

    (2012)
  • S. Huang et al.

    Decelerated genome evolution in modern vertebrates revealed by analysis of multiple lancelet genomes

    Nat. Commun.

    (2014)
  • Cited by (2)

    1

    Yi Shi and Zhaoyu Fan contribute equally to this work.

    View full text