A gene seems to be lost in chicken - is it really true?

Plg-RKT or Plasminogen Receptor K terminal was first identified a decade ago (in the year 2010) based on a proteomics study. Given its recent discovery, the number of research papers focused solely on this protein are limited. List of the papers dealing with the discovery and functional characterization of this protein as well as its relevance to understanding the healthy and disease state of the body are provided in the references. Prior to its characterization, Plg-RKT was known as C9orf46 due to its presence on human chromosome 9 (ORF 46). The original discovery of the role of Plg-RKT and most of the subsequent work related to this protein emanate from the lab of Lindsey Miles (Professor of Cell and Molecular Biology) at The Scripps Research Institute, La Jolla, CA. The 2010 paper notes

“Our isolation of peptides corresponding to C9orf46 homolog is, to our knowledge, the first demonstration of the existence of this protein. We have designated the protein, Plg-R_KT, to indicate a plasminogen receptor with a C-terminal lysine and having a transmembrane domain.”

Presence of the C-terminal lysine in this protein seems to be highly conserved across mammals and birds. This lysine residue is exposed on the cell surface and is recognized by plasminogen. The known functions of the Plg-RKT gene can be summarized as follows:

1. Regulation of macrophage phenotype
2. Mammary development and lactation
3. Regulation of efferocytosis
4. Metabolic homeostasis and adipose function
5. Mediation of Lipoprotein(a) endocytosis
6. Regulation of cell surface plasminogen activation

Given the evidence for multiple important functional roles of this gene, it seems unlikely that this gene is dispensable. Several other receptors for plasminogen do exist and could potentially play a compensatory role. The orthologs of Plg-RKT are well conserved and a 1 to 1 ortholog (CG13404 (FBgn0030559)) is annotated in Drosophila melanogaster. A recent pre-print implicates this orthologous gene in Coxiella burnetii Infection susceptibility in Drosophila melanogaster based on a GWAS study that relies on DGRP. Although no ortholog is annotated in yeast, two homologs (tag-280 (WBGene00044322) and tag-281 (WBGene00044323)) are annotated in C. elegans and remain uncharacterised.

To further identify potential gene losses in chicken, we obtained a list of genes that are co-expressed with Plg-RKT in human samples or otherwise known to interact with Plg-RKT and evaluated whether their orthologs are present in chicken. 

Sl. No	Human Gene stable ID	Gene name	Chicken ortholog	Remark
1	ENSG00000062038	CDH3	ENSGALG00000051984	Pseudogene annotation on ensemble but annotated mRNA with ORF at KY120273.1
2	ENSG00000137975	CLCA2	ENSGALG00000050155	Ortholog found
3	ENSG00000149547	EI24	ENSGALG00000038097	Ortholog found
4	ENSG00000126749	EMG1	ENSGALG00000014568	Ortholog found
5	ENSG00000068438	FTSJ1	Is this lost ??	Chicken Chr 12 and Chr 13 breakpoint
6	ENSG00000189280	GJB5	ENSGALG00000054289	Ortholog found
7	ENSG00000108010	GLRX3	ENSGALG00000010464	Ortholog found
8	ENSG00000196743	GM2A	ENSGALG00000027534	Ortholog found
9	ENSG00000138271	GPR87	ENSGALG00000010377	Ortholog found
10	ENSG00000113161	HMGCR	ENSGALG00000014948	Ortholog found
11	ENSG00000053747	LAMA3	ENSGALG00000015056	Ortholog found
12	ENSG00000172172	MRPL13	ENSGALG00000041863	Ortholog found
13	ENSG00000131467	PSME3	ENSGALG00000002937	Ortholog found
14	ENSG00000087494	PTHLH	ENSGALG00000017295	Ortholog found
15	ENSG00000176225	RTTN	ENSGALG00000013745	Ortholog found
16	ENSG00000104549	SQLE	ENSGALG00000036915	Ortholog found
17	ENSG00000056972	TRAF3IP2	ENSGALG00000015026	orthology not annotated
18	ENSG00000087245	MMP2	ENSGALG00000003580	Ortholog found
19	ENSG00000100985	MMP9	ENSGALG00000006992	Ortholog found

Most of the above genes have clear 1 to 1 orthologs in chicken. The origin and diversification of the plasminogen activation system has been explored by looking at homologs of 15 genes consisting of the following groups:

• PLG, HGF and MST-1
• HABP2, HGFAC, tPA and uPA
• SERPINE1 (PAI-1), SERPINE2, SERPINE3 and SERPINI1
• PAI-2
• VTN
• 3LU and uPAR

When the orthologs of these genes are searched in chicken, we again find most of them. The exceptions are PLAUR and SERPINE1. Prior work has suggested these genes are lost in chicken. In addition, to these loss events, we see duplication of PLAU and PLG like loci. The potential loss of PLAUR could be interesting as PLAUR is known to interact with PLG. 

Gene stable ID	Gene name	Chicken gene stable ID
ENSG00000173531	MST1	ENSGALG00000002722
ENSG00000122861	PLAU *	ENSGALG00000050317
ENSG00000122861	PLAU *	ENSGALG00000046993
ENSG00000148702	HABP2	ENSGALG00000008905
ENSG00000163536	SERPINI1	ENSGALG00000009470
ENSG00000122194	PLG *	ENSGALG00000028886
ENSG00000122194	PLG *	ENSGALG00000004293
ENSG00000135919	SERPINE2	ENSGALG00000005135
ENSG00000011422	PLAUR
ENSG00000104368	PLAT	ENSGALG00000003709
ENSG00000253309	SERPINE3	ENSGALG00000017017
ENSG00000109758	HGFAC	ENSGALG00000015623
ENSG00000019991	HGF	ENSGALG00000033974
ENSG00000106366	SERPINE1
ENSG00000109072	VTN	ENSGALG00000003589

We next compiled the list of all the plasminogen receptors from previous reviews.

Sl No	Gene stable ID	Gene symbol	Chicken gene stable ID	Gene name	Remarks
1	ENSG00000074800	ENO1	ENSGALG00000002377	alpha-enolase	First plasminogen receptor to be identified. See: Activation of plasminogen into plasmin at the surface of endothelial microparticles: a mechanism that modulates angiogenic properties of endothelial progenitor cells in vitro (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2495018/)
2	ENSG00000189403	HMGB1	ENSGALG00000042875	Amphoterin
3	ENSG00000197747	S100A10	ENSGALG00000028774	P11
4	ENSG00000107020	PLGRKT	Is this really lost?	Plg-RKT
5	ENSG00000182718	ANXA2	ENSGALG00000003770	Annexin A2
6	ENSG00000170421	KRT8	ENSGALG00000050400	Cytokeratin 8	Orthology not annotated
7	ENSG00000005961	ITGA2B	ENSGALG00000054766	Integrin Alpha-IIb/beta-3
8	ENSG00000138448	ITGAV	ENSGALG00000002655	Integrin AlphaVbeta3
9	ENSG00000169896	ITGAM	Orthologs found in lizard and alligator but not in birds. Duplication of ITGAX (see: Structural analysis of the CD11b gene and phylogenetic analysis of the alpha-integrin gene family demonstrate remarkable conservation of genomic organization and suggest early diversification during evolution.(https://www.jimmunol.org/content/150/2/480.long))	Integrin Subunit Alpha M	Integrin αMβ2 Orchestrates and Accelerates Plasminogen Activation and Fibrinolysis by Neutrophils (https://www.jbc.org/content/279/17/18063.long)
10	ENSG00000160255	ITGB2	ENSGALG00000007511	Integrin subunit beta 2
11	Histone genes occur in cluster and all copies retain high levels of sequence similarity. See: Molecular Evolution of the Nontandemly Repeated Genes of the Histone 3 Multigene Family (https://academic.oup.com/mbe/article/19/1/68/1066713)			Histone 2B	Phosphatidylserine as an anchor for plasminogen and its plasminogen receptor, Histone H2B, to the macrophage surface (https://onlinelibrary.wiley.com/doi/full/10.1111/j.1538-7836.2010.04132.x)

The Immunogenetics journal has previously (2019 [Convergent inactivation of the skin-specific C-C motif chemokine ligand 27 in mammalian evolution (https://link.springer.com/article/10.1007/s00251-019-01114-z)] and 2018 [Cetacea Are Natural Knockouts for IL20 (https://pubmed.ncbi.nlm.nih.gov/29998404/)]) published gene loss stories in cetaceans. However, both IL20 and CCL27 are well studied genes and the observed loss spanned several species including independent losses. The authors could also provide a fairly convincing explanation for why these genes were lost in cetacean species and provide evidence from re-sequencing datasets and RNA-seq experiments. Loss of NLRC4 and NAIP in pigs was reported [Pig lacks functional NLRC4 and NAIP genes (https://link.springer.com/article/10.1007/s00251-016-0955-5)] in 2017. Being a domesticated species, changes in the immune repertoire of the pig has implications for the pork industry. Interestingly, this paper makes a reference to the lack of RIG-I in chicken and how the immune response is different because of this. 

Given all this background information we wanted to make sure we provided enough evidence for the pseudogenisation of PLGRKT to convince the reviewers. The recently published online PseudoChecker tool failed to find the remnants of the PLGRKT gene. Neither was it able to find the intact gene in Duck when the human exons and CDS were used as the reference. So PLGRKT can be added to the list of less than 5% genes that PseudoChecker is supposedly unable to find. Fortunately, we have been told that real lossomicists (scientists whose specialty is finding gene loss events) use exon by exon tblastx followed by careful scrutiny to prove gene loss. So we did this and find very clear evidence for the existence of exon-3 remains and largely intact exon-4 in chicken. Side by side comparison with results with duck cDNA are provided here: https://github.com/ceglab/PLGRKT/tree/master/tblastx. So all this and the evidence presented by Sharma et. al., suggests this genes is truly lost in chicken. 

In addition to the work done by the Miles lab, recently published papers are from the University of Otago (see talk describing the work here (PLGRKT starts around 30 minutes into the video): https://www.youtube.com/watch?v=0bpNZSZdeQU) and Medical University of Vienna (see video here: https://www.youtube.com/watch?v=xjPmTDkhWr8).

 References

1.      Plasminogen and the Plasminogen Receptor, Plg-RKT, Regulate Macrophage Phenotypic, and Functional Changes (https://www.frontiersin.org/articles/10.3389/fimmu.2019.01458/full)

2.      The Plasminogen Receptor, Plg-RKT, and Macrophage Function (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3484331/)

3.      The Plasminogen Receptor, Plg-RKT, is Essential for Mammary Lobuloalveolar Development and Lactation (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5965281/)

4.      The Novel Plasminogen Receptor, Plasminogen ReceptorKT (Plg-RKT), Regulates Catecholamine Release (https://www.jbc.org/content/286/38/33125.full)

5.      Plasminogen receptors and their role in the pathogenesis of inflammatory, autoimmune and malignant disease (https://onlinelibrary.wiley.com/doi/pdf/10.1111/jth.12064)

6.      Deficiency of Plasminogen Receptor, Plg-RKT, Causes Defects in Plasminogen Binding and Inflammatory Macrophage Recruitment in vivo (https://pubmed.ncbi.nlm.nih.gov/27714956/)

7.      Plasminogen and the Plasminogen receptor, Plg-RKT, regulate efferocytosis and macrophage reprogramming (https://www.fasebj.org/doi/abs/10.1096/fasebj.2018.32.1_supplement.280.4)

8.      The Plasminogen Receptor, Plg-RKT, Regulates Metabolic Homeostasis and Promotes Healthy Adipose Function (https://www.ahajournals.org/doi/abs/10.1161/circ.134.suppl_1.19088)

9.      Proteomics-based discovery of a novel, structurally unique, and developmentally regulated plasminogen receptor, Plg-RKT, a major regulator of cell surface plasminogen activation (https://ashpublications.org/blood/article/115/7/1319/26700/Proteomics-based-discovery-of-a-novel-structurally)

10.  Regulation of Macrophage Migration by a Novel Plasminogen Receptor Plg-R KT (https://pubmed.ncbi.nlm.nih.gov/21940822/)

11.  New Insights Into the Role of Plg-RKT in Macrophage Recruitment (https://pubmed.ncbi.nlm.nih.gov/24529725/)

12.  Plasminogen Receptors: The First Quarter Century (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938387/)

13.  New Insight on the Role of Plasminogen Receptor in Cancer Progression (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4521684/)

14.  Plasminogen Receptors in Human Malignancies: Effects on Prognosis and Feasibility as Targets for Drug Development (https://pubmed.ncbi.nlm.nih.gov/31755385/)

15.  Differential expression of Plg-RKT and its effects on migration of proinflammatory monocyte and macrophage subsets (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688429/)

16.  Recycling of Apolipoprotein(a) After PlgRKT-Mediated Endocytosis of Lipoprotein(a) (https://www.ahajournals.org/doi/full/10.1161/circresaha.116.310272)

17.  Natural Genetic Variation in Drosophila melanogaster Reveals Genes Associated with Coxiella burnetii Infection  (https://www.biorxiv.org/content/10.1101/2020.05.21.109371v1.full)

18. Origin and diversification of the plasminogen activation system among chordates (https://bmcevolbiol.biomedcentral.com/articles/10.1186/s12862-019-1353-z)