FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells

Abstract

Background: Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line.

Results: One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20) with three members (FAM20A, FAM20B and FAM20C) in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c) were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members.

Conclusions: The FAM20 family represents a new family of secreted proteins with potential functions in regulating differentiation and function of hematopoietic and other tissues. The Fam20a mRNA was only expressed during early stages of hematopoietic development and may play a role in lineage commitment or proliferation. The expansion in gene number in different species suggests that the family has evolved as a result of several gene duplication events that have occurred in both vertebrates and invertebrates.

Figure 1

Characterization of the full length mouse 1623 (Fam20a) cDNA and genomic sequence. A. The full length cDNA derived from the original RDA clone was isolated using a combination of 5' and 3' rapid amplification of cDNA ends (RACE) procedures, comparisons to public databases, and amplification of putative full length clones by PCR. The full open reading frame was 1623 bp in length and encoded a 541 amino acid protein. The locations of regions conserved within the subsequently identified FAM20 family are indicated using underlines. Eight cysteine residues that are also conserved within the family are indicated in bold and four putative N-glycosylation sites are indicated in red type. B. The distribution of the 11 exons of the mouse Fam20a gene is shown with the exons indicated using numbers. A consensus polyadenylation signal is located downstream of the terminal exon. C. The sizes of the 11 exons and 10 introns of the Fam20a gene are shown.

Figure 2

Evolutionary distribution of FAM20 gene number. An idealized evolutionary tree (modified from [10]) is shown with the number of FAM20 genes identified in several genomes as described in the text. The gene numbers are supportive of a single gene duplication event occurring in invertebrates (at least in insects) and multiple gene duplication events occurring in higher vertebrates.

Figure 3

Assignment of FAM20 family members to subfamilies. A. Exon size and distribution of mammalian FAM20 members. The exons within each FAM20 gene in human, mouse and rat are indicated with the number of base pairs indicated within each exon. The sizes of exons that differ in size from the FAM20A genes are indicated. B. A dendrogram showing the relationships between FAM20 proteins from human (Hs), mouse (Mm), rat (Rn), Fugu rubripes (Fr), Danio rerio (Dr), D. melanogaster (Dm), A. gambiae (Ag), C. intestinalis (Ci) and C. elegans. The accession numbers of the cDNA sequences from which each protein sequence was derived are shown in parentheses except in the case of the mosquito family members where the accession number is used as the gene/protein name. Accession numbers for zebrafish peptide sequences are listed in Table 1. The FAM20 nomenclature has not been extended to the invertebrate sequences and the previous gene names have been used for Drosophila and C. elegans family members. The subfamily assignment of each family member is shown on the right. C. Exon number and size distribution within Fugu Fam20 members. The accession number of each sequence within the Third Party Annotation database is shown at left and family assignment based on dendrogram position and exon distribution is shown on the right.

Figure 4

Sequence alignment of FAM20A protein sequences. The complete protein sequences of FAM20A members were compared using the AlignX component of the VectorNTI sequence analysis suite of programs. Identical amino acids are outlined in yellow, and similar residues are indicates in light blue. Conserved regions 1, 2 and 3 are underlined (see below). Gaps are indicated with dashes and the sequences are from human (H), mouse (M), rat (R) and puff erfish (F).

Figure 5

Sequence alignment of FAM20B protein sequences. The complete protein sequences of FAM20B members are presented as described in figure 4. The sequences are from human (H), mouse (M), rat (R), pufferfish (F), zebrafish (D) and C. intestinalis (Ci).

Figure 6

Sequence alignment of FAM20C protein sequences. The complete protein sequences of FAM20C members are presented as described in figure 4. The sequences are from human (H), mouse (M), rat (R), pufferfish (Fcl-4) and zebrafish (Dcl-3).

Figure 7

Schematic representation of the structural features of FAM20 family members. A. Structural features of FAM20A showing domains and residues conserved within the entire family. Key: SS: signal sequence; CCD: conserved C-terminal domain; CR: conserved region; Cys: cysteine residues conserved within CCD (indicated with asterisk). B. Consensus sequences were derived for CR1, CR2 and CR3 using a global comparison of all the family members listed in Tables 1 and 2. Residues that are invariant or only differ in one sequence are indicated in bold. Non-conserved residues are indicated with an x and positions with more than one common residue are shown below the main sequence.

Figure 8

Fam20a is a secreted protein. COS-1 cells were transfected with either an empty expression vector (-) or one encoding mouse Fam20a with a C-terminal myc epitope tag and proteins were isolated from either the medium (panel A) or the cells (panel B). The proteins were analyzed by immunoblotting using a Myc tag-specific antiserum. Samples in lanes 2 and 4 of each blot were pre-treated with protein N-glycosidase prior to analysis to remove glycosyl groups. A recombinant form of Fam20a synthesized in rabbit reticulocyte lysates (TnT) was included on each gel as a size marker. The position of glycosylated and deglycosylated Fam20a is indicated using arrowheads and cross reacting material detected in the medium is indicated using asterisks. The location of molecular size markers is shown on the left of each gel. C. Protein samples from the medium of transfected cells that were untreated or treated with Brefeldin A were analyzed by immunoblotting using the Myc tag-specific antiserum. As Brefeldin A was resuspended in DMSO, the untreated cells were exposed to DMSO alone as a vehicle control. The amount of Fam20a detected in the medium of Brefeldin A treated cells was consistently lower than that observed in untreated cells (indicated using an arrowhead).

Figure 9

Secretion of Fam20a requires an intact signal sequence. A. Schematic representation of the putative signal sequence of Fam20a. The predicted cleavage site is indicated with a red arrowhead. The two amino acid substitutions introduced in the SSmut construct and the sequence remaining in the Δ23 mutant construct are shown. B. Immunoblot analysis of Fam20a and Fam20a(Δ23) protein levels in transfected COS-1 cells. The position of the glycosylated form of Fam20a (which is absent in Fam20a(Δ23) transfected cells is indicated with an arrowhead. C. Fluorescence images of COS-1 cells expressing either Fam20a-GFP or Fam20a(Δ23)-GFP. The wild type protein was observed within the cytoplasm, predominantly in a structure that is likely to be the ER. The mutant protein was primarily localized to the nucleus. D. Immunofluorescence images of Fam20a and Fam20a (SSmut) proteins as detected by antiserum directed against the C-terminal Myc epitope. The wild type protein was again detected in the ER and the mutant protein primarily in the nucleus. The cells have been counterstained with DAPI to delineate the nucleus.

Figure 10

RT-PCR analysis of mouse Fam20 mRNA levels during differentiation of EML and MPRO cells. Total RNAs were prepared from EML (panel A) or MPRO (panel B) cells at the indicated timepoints during myeloid and granulocytic differentiation. cDNAs prepared from each sample were amplified using primer pairs specific to each mouse family member. The PCR products were analyzed by agarose gel electrophoresis and stained using Gelstar SYBR Green DNA stain. GAPDH was used as a loading control.

Figure 11

RT-PCR analysis of human FAM20 mRNA levels in human tissues. A panel of commercially available human cDNAs prepared from the indicated tissues was analyzed by PCR using primer pairs specific for each of the human FAM20 family members. GAPDH was again used as a loading control although large variations were observed in the GAPDH signal in the different tissues.