Phylogenetic analysis of the MS4A and TMEM176 gene families

Abstract

Background: The MS4A gene family in humans includes CD20 (MS4A1), FcRbeta (MS4A2), Htm4 (MS4A3), and at least 13 other syntenic genes encoding membrane proteins, most having characteristic tetraspanning topology. Expression of MS4A genes is variable in tissues throughout the body; however, several are limited to cells in the hematopoietic system where they have known roles in immune cell functions. Genes in the small TMEM176 group share significant sequence similarity with MS4A genes and there is evidence of immune function of at least one of the encoded proteins. In this study, we examined the evolutionary history of the MS4A/TMEM176 families as well as tissue expression of the phylogenetically earliest members, in order to investigate their possible origins in immune cells.

Principal findings: Orthologs of human MS4A genes were found only in mammals; however, MS4A gene homologs were found in most jawed vertebrates. TMEM176 genes were found only in mammals and bony fish. Several unusual MS4A genes having 2 or more tandem MS4A sequences were identified in the chicken (Gallus gallus) and early mammals (opossum, Monodelphis domestica and platypus, Ornithorhyncus anatinus). A large number of highly conserved MS4A and TMEM176 genes was found in zebrafish (Danio rerio). The most primitive organism identified to have MS4A genes was spiny dogfish (Squalus acanthus). Tissue expression of MS4A genes in S. acanthias and D. rerio showed no evidence of expression restricted to the hematopoietic system.

Conclusions/significance: Our findings suggest that MS4A genes first appeared in cartilaginous fish with expression outside of the immune system, and have since diversified in many species into their modern forms with expression and function in both immune and nonimmune cells.

Figure 1. Predicted topology and length of human MS4A and TMEM176 proteins.

Line diagrams are drawn to scale. Thick lines represent transmembrane domains. Thin lines represent intracellular and extracellular regions. N termini are predicted to localize in the cytoplasm.

Figure 2. Phylogram of MS4A and TMEM176 families.

Using the multiple sequence alignment shown in Supplementary Figure S1, a phylogeny was generated of all sequences listed in Supplementary Table S2, using a sequence from S. purpuratus to root the tree (SP_out). The expanded phylogenetic tree with bootstrap values is shown in supplementary Figure S2.

Figure 3. Tandem MS4A genes in G. gallus, M. domestica and O. anatinus.

The long MS4A protein sequences were cut into their subunit components and renamed with the suffix A through F to denote their respective position relative to the amino terminus. Alignment and phylogeny of these sequences was performed using ClustalX. The detailed alignments are shown in supplementary Figure S3. A. An unrooted phylogeny of the MS4A subunit sequences compared to full length human MS4A sequences. B. A schematic representation of the sequence alignments was generated using Genedoc. Shading indicates the degree of sequence conservation. Black shading indicates that the amino acid is conserved; grey indicates partial conservation and white indicates there is little to no sequence conservation at the indicated position.

Figure 4. Tissue expression of MS4A17A.17 in D. rerio.

Whole mount embryos (upper panels) and coronal sections (lower panels) of in situ hybridization of MS4A17A.17 probe in zebrafish 96 hours post fertilization embryo. Probe hybridized to the dorsal pharynx (arrow a in upper and lower left panels), an unidentified neuron (arrow c in upper right panel), and the mesenchyme surrounding the kidney glomerulus (arrow b in the upper left, lower middle and lower right panels).

Figure 5. Tissue expression of MS4A genes in S. acanthus.

Reverse transcriptase PCR was performed on RNA isolated from the tissues indicated, using primers to amplify each of the 3 distinct MS4A genes identified in the S. acanthus EST database. CD79a is a B lymphocyte expressed gene used to detect the presence of blood cells in each tissue or organ examined. Beta actin was used as a standardizing gene.