cDNA and Gene Structure of MytiLec-1, A Bacteriostatic R-Type Lectin from the Mediterranean Mussel (Mytilus galloprovincialis)

Abstract

MytiLec is an α-d-galactose-binding lectin with a unique primary structure isolated from the Mediterranean mussel (Mytilus galloprovincialis). The lectin adopts a β-trefoil fold that is also found in the B-sub-unit of ricin and other ricin-type (R-type) lectins. We are introducing MytiLec(-1) and its two variants (MytiLec-2 and -3), which both possess an additional pore-forming aerolysin-like domain, as members of a novel multi-genic "mytilectin family" in bivalve mollusks. Based on the full length mRNA sequence (911 bps), it was possible to elucidate the coding sequence of MytiLec-1, which displays an extended open reading frame (ORF) at the 5' end of the sequence, confirmed both at the mRNA and at the genomic DNA sequence level. While this extension could potentially produce a polypeptide significantly longer than previously reported, this has not been confirmed yet at the protein level. MytiLec-1 was revealed to be encoded by a gene consisting of two exons and a single intron. The first exon comprised the 5'UTR and the initial ATG codon and it was possible to detect a putative promoter region immediately ahead of the transcription start site in the MytiLec-1 genomic locus. The remaining part of the MytiLec-1 coding sequence (including the three sub-domains, the 3'UTR and the poly-A signal) was included in the second exon. The bacteriostatic activity of MytiLec-1 was determined by the agglutination of both Gram-positive and Gram-negative bacteria, which was reversed by the co-presence of α-galactoside. Altogether, these data support the classification of MytiLec-1 as a member of the novel mytilectin family and suggest that this lectin may play an important role as a pattern recognition receptor in the innate immunity of mussels.

Keywords: MytiLec-1; Mytilus galloprovincialis; R-type lectin; bacteriostatic activity; cDNA; gene; innate immunity; mRNA-sequence; mytilectin family.

Figure 1

cDNA sequence and deduced amino acid sequence of MytiLec-1. Bold and italic numbers indicate nucleotides and amino acids, respectively. ³⁸Met (yellow background) correspond to the first amino acidic residue of the mature MytiLec-1 polypeptide. The alternative N-terminal extension sequence is underlined. The asterisk indicates the stop codon.

Figure 2

Deduced amino acid sequences of the three members of the mytilectin family in M. galloprovincialis (MytiLec-1, -2, and -3). Alternative N-terminal extension (orange), first sub-domain (blue), second sub-domain (green), third sub-domain (pink), and aerolysin-like domain (gray) are shown here. Asterisks denote amino acids essential to ensure the sugar-binding properties [10]. Bold characters within these essential amino acids indicate amino acids that are conserved compared to MytiLec-1.

Figure 3

Gene structure of MytiLec-1. The gene and its promoter are indicated as blue and red arrows, respectively. Two exons of the lectin are indicated as a broken green arrow. The coding sequence, including the N-terminal alternative extension, is indicated by a yellow arrow, and the position ³⁸Met is indicated by a black arrow. The RNA-seq mapping graphs obtained from digestive gland and gills clearly mark the location of exon/intron junctions.

Figure 4

Gene expression of MytiLec-1 in different adult mussel tissues, calculated based on RNA-seq data. Expression levels are given as RPKM (read per kilobase per million mapped reads). HEM: hemocytes; DG: digestive gland; GIL: gills; PAM: posterior adductor muscle; AMA: anterior muscle; MMA: mid mantle; and PMA: posterior mantle.

Figure 5

Agglutination of E. coli by MytiLec-1. MytiLec-1 (20 μg/mL in (A), (C), and (D)) was applied to the bacteria with 50 mM of melibiose (Galα1-6Glc) and lactose (Galβ1-4Glc), shown in (C) and (D), respectively. (B) (PBS) is the negative control without lectin. The black bar indicates a scale of 25 μm.