Transmembrane protein 211 (TMEM211,bA9F11.1,Q6ICI0,LHFLP7) is a tetraspan membrane protein under the LHFPL subfamily. It primarily plays a role in the perception of sound but may have secondary roles in insulin signaling. It is encoded by the TMEM211 gene and is found in almost all animals.
Expression and localization Human TMEM211 RNA is expressed in relatively low levels, but displays clear spikes in the tissues of the brain, stomach, lungs, breasts, ovaries, prostate, trachea, and salivary glands. In the fetus, TMEM211 RNA is again expressed in the brain and stomach, but is also expressed in the intestines. TMEM211 is also known to be expressed in skin, in significantly greater amounts in sun-exposed skin than in non-sun-exposed skin, and overexpressed in triple-negative breast cancer. In a compendium of healthy canine tissues, TMEM211 displayed the highest expression in the pancreas (Figure 1).“TMEM211 - Compendium of normal tissues.” 81313044 - GEO Profiles - NCBI. (2021). https://www.ncbi.nlm.nih.gov/geoprofiles/81313044 Within the pancreas, TMEM211 displays highly biased abundance in the islets of Langerhans, and is absent from other pancreatic tissue(Figure 2).Anti-TMEM211 Antibody (HPA066784) - Atlas Antibodies. https://www.atlasantibodies.com/products/antibodies/primary-antibodies/triple-a-polyclonals/tmem211-antibody-hpa066784/ TMEM211 is localized to the plasma membrane as a result of its four transmembrane helices.
Despite the protein's localization to the uterus, ovaries, and breast milk, oral administration of estradiol to menopausal women does not produce a significant change in the level of TMEM211 expression. However, it did significantly reduce the variability of TMEM211 between samples, indicating that estrogen does exert a controlling effect on TMEM211, likely through an indirect mechanism (Figure 3). TMEM211 was also shown to be expressed in higher levels in obese individuals (Figure 4). This result may be explained by the finding that obesity, especially non-diabetic obesity, is correlated with an increase in both the volume and number of islets of Langerhans. Alzheimer's patients likewise displayed higher TMEM211 expression compared to non-Alzheimer's individuals, as did women when compared to men (Figure 5).
Translations and homologs
Figure 6 displays select regions of interest of the TMEM211 human protein sequence. Most notably, it shows the location and presence of the transmembrane regions and the exon boundary. The RNA transcript is made of four exons, but only two of these exons contain coding sequence. Uniquely, the protein has two start codons, both of which can begin translation and lead to protein products. This is the main cause of TMEM211 isoforms.
Ortholog space
TMEM211 is present in the majority of animal species, but does not exist outside of animals (Figure 7). The oldest group of organisms where the gene is found ubiquitously is anemones, which diverged from humans 824 million years ago. The gene is not found in organisms that diverged 934 million years ago, indicating that the gene is between 824 and 934 million years old. Following evolution, the gene is then found in fish, amphibians, reptiles, birds, and mammals. However, there is one clade with a different pattern of expression, arthropods. The gene is readily found in crustaceans, but is missing from all other arthropods. For unknown reasons, marine arthropods maintain the TMEM211 gene while terrestrial arthropods have lost it.
There is one possible exception to this, the White Butterfly Parasite. This gene is found by BLAST when searching for human TMEM211, and searching for White Butterfly Parasite's TMEM211 sequence did return results in other parasitic wasps, indicating that this was not likely a sequencing or contamination error. This small clade may have preserved the gene while the rest of the terrestrial arthropods lost it. However, the TMEM211 genes found within this clade are significantly less similar than every other organism with the same ancestral distance from humans. While the gene may be an ortholog, it also may be serving a new function for this group of wasps, which is why this clade retained the gene while other insects lost it. This would also explain the higher percent divergence. Similarly, it is possible that this group does not express the gene, which would mean that no evolutionary forces work against its mutation, allowing it to change faster than in organisms where the gene must serve as a template for a functional protein. Finally, it has been proven that the caterpillar species that this group of wasps preys upon uses an entomopathogenic defense mechanism. This virus does not cause illness to the caterpillar, but does infect the wasp eggs and wasp larvae and has been shown to cause horizontal gene transfer. It is easily plausible that the TMEM211 gene was given to this subset of wasps by one of these viruses. Then, as the gene was artificially created in the genome and does not serve a real purpose, it mutates freely, leading to the observed divergence that is higher than expected. This explanation is supported by the fact that the intron that separates the two halves of the protein is missing. Alternatively, the BLAST result might be misleading, and what is flagged as TMEM211 is actually a member of the paralogous LHFPL family, which is known to be found in both wasps and caterpillars, and includes members that do not share the TMEM211 intron.
Sequence alignments The MSA of distant orthologs highlights several conserved regions (Figure 8). However, the human gene does not partake in the majority of this conservation, nor do the other mammals. This is consistent with the protein's presence in breastmilk; non-mammalian species do not have breastmilk, and thus the protein may have slightly different functions in mammals and non-mammals. Much of the protein's identity that is found to match appears scattered, but there is one clear region of highest conservation. In humans, this region spans amino acids 132–150. The other two regions with high conservation shared by humans span amino acids 59-71 and 110–117. There are only six amino acids with complete conservation across all species, Cys27, Gly 65, Gln100, Pro115, Cys127, Cys138. Half of the completely conserved amino acids are cysteine, thus cysteine's sulfhydryl group and ability to form disulfide bridges and covalent bonds may be important to the protein's structure and/or function.
Overall, the amino acids with the highest conservation in respect to the human sequence are tyrosine (81%) and tryptophan (72%). These two amino acids are vital to transmembrane proteins as they can interact with both the hydrophobic membrane and the aqueous environment inside or outside of the cell, and thus, it is logical that these amino acids are highly conserved. Looking at the mammals’ alignment, the majority of the protein is conserved (Figure 9). In fact, every protein that is wholly unconserved is found embedded within the membrane, where exact identity likely matters less to the function of the protein. This pattern remains true in the broader MSA of orthologs, where the regions of least conservation are mostly inside the membrane. While keeping this conservation, TMEM211 has evolved and mutated with the organisms that carry it. The divergence of each organism's gene follows normal phylogeny (Figure 10).
Conserved regions There is evidence that suggests that the transmembrane segments are conserved to a lesser degree than are the other segments of the protein, a notion supported by the frequent swapping of small, nonpolar amino acids within these regions. To test this hypothesis, Shannon Variability bits were computed for each position within the protein (Figure 11). While this correlation is indeed observed, the relationship is not great enough to be conclusive. Using the Shannon bits, Figure 12 converts the annotated conceptual translation to an annotated representation that better highlights the proximity of the conserved regions and amino acids in three-dimensional space. The first and third conserved regions are located adjacent to one another on the extracellular side of the protein, possibly creating an active site. The orientation of the transmembrane domains also brings several cysteines into close proximity, allowing them to form disulfide bridges.
Paralogs
A BLAST query of the human TMEM211 sequence within the human genome returns no results. This would seem to indicate that humans have no paralog. However, submitting non-mammalian TMEM211 sequences as queries returns Homo sapiens Lipoma high-mobility group I C protein fusion partner-like tetraspan subfamily member 3 protein isoform 2 (LHFPL3). Additionally, BLAST queries of non-mammalian TMEM211 sequences searching within those organisms’ genomes return numerous results within the LHFPL family. This protein is similar to TMEM211 in the fact that it is a tetraspan membrane protein. To evaluate whether these four transmembrane regions are the cause for high similarity, a global alignment between human TMEM211 and human LHFPL3 was created (Figure 13). The similarity does not appear confined to the transmembrane regions, indicating that these two proteins may be paralogous. Additionally, 4 of the 6 amino acids completely conserved in TMEM211 orthologs are aligned between TMEM211 and LHFPL3, furthering the evidence that these proteins are paralogs (p<.01).
The LHFPL family is of known function; members of the family aid protein binding in the brain and are vital to the perception of sound. Knockouts or loss of function mutations to these family members cause complete or partial deafness in humans and mice that is then inherited in an autosomal recessive pattern. Other mutations to LHFPL have been linked to tumors, autism, Alzheimer's, and other neurological conditions. The conserved cysteine residues in TMEM211 indicate that this could be a possible function for TMEM211 as well. The conserved regions that align in three-dimensional space may form a binding site that uses the conserved cysteine residues to recognize and bind substrates. Similar to TMEM211, the LHFPL family members show biased expression in the brain and salivary glands. LHFPL was not shown to be expressed in the pancreas, but per the aforementioned discussion, exclusion of the islets of Langerhans from pancreatic samples would lead to this result. LHFPL RNA, like TMEM211, was present in lower levels in the GI tract, kidneys, prostate, thyroid, and sex organs. LHFPL family members have been studied more so than TMEM211, and protein abundance data shows that each family member, with the exception of LHFPL2, displays its highest abundance level in healthy tissues in the brain. While still found in brain tissue at higher concentrations than the majority of other brain proteins, LHFPL2 displays an abundance in platelets that is 60 times greater, in the top 10% of platelet proteins. Each family member also displayed heightened abundance in lung cancer cell lines, as did TMEM211. Several LHFPL family members were also found in breastmilk, but in greatly lower quantities than TMEM211. Despite the association with hearing and sound perception, neither TMEM211 or any LHFPL family members show a pattern of localization biased to the thalamus, temporal lobe, or auditory cortex.TMEM211 protein expression summary - The Human Protein Atlas. (2021). Retrieved from https://www.proteinatlas.org/ENSG00000206069-TMEM211Allen Institute for Brain Science (2004). Allen Brain Atlas.