Name: creating and applying a reference to facilitate the discussion and classification of proteins in a diverse group
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Ongoing tardigrade antimicrobial peptide research is supported by intramural funding from the Midwestern University Office of Research and Sponsored Programs (ORSP). The ORSP had no role in study design, data collection, analysis, interpretation, or manuscript preparation.

1. Determine the Defining Features of the Protein Group of Interest
<ol>
	<li>Consult previous publications to determine if there is a consensus regarding the features that are necessary to be considered part of the group. Take note of any inconsistencies or differences in opinion between research groups, and include characteristics that may serve to differentiate one subgroup from another.</li>
	<li>If previous literature does not address defining characteristics, use sequences that are considered representative of th...

<ol>
	<li>Matsuyama, K., Natori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Three+Antibacterial+Proteins+from+the+Culture+Medium+of+NIH-Sape-4,+an+Embryonic+Cell+Line+of+Sarcophaga+peregrina.">Purification of Three Antibacterial Proteins from the Culture Medium of NIH-Sape-4, an Embryonic Cell Line of Sarcophaga peregrina.</a> J Biol Chem. 263 (32), 17112-17116 (1988).</li><li>Lambert, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+immunity:+Isolation+from+immune+blood+of+the+dipteran+Phormia+terranovae.+of+two+insect+antibacterial+peptides+with+sequence+homology+to+rabbit+lung+macrophage+bactericidal+peptides.">Insect immunity: Isolation from immune blood of the dipteran Phormia terranovae. of two insect antibacterial peptides with sequence homology to rabbit lung macrophage bactericidal peptides.</a> PNAS. 86 (262-266), (1989).</li><li>Dimarcq, J. -. L., Bulet, P., Hetru, C., Hoffmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cysteine-rich+antimicrobial+peptides+in+invertebrates.">Cysteine-rich antimicrobial peptides in invertebrates.</a> Biopolymers. 47, 465-477 (1998).</li><li>Shafee, T. M. A., Lay, F. T., Hulett, M. D., Anderson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Defensins+Consist+of+Two+Independent,+Convergent+Protein+Superfamilies.">The Defensins Consist of Two Independent, Convergent Protein Superfamilies.</a> Mol Biol Evol. 33 (9), 2345-2356 (2016).</li><li>Zhu, S., Gao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nematode-derived+drosomycin-type+antifungal+peptdies+provide+evidence+for+plant-to-ecdysozoan+horizontal+transfer+of+a+disease+resistance+gene.">Nematode-derived drosomycin-type antifungal peptdies provide evidence for plant-to-ecdysozoan horizontal transfer of a disease resistance gene.</a> Nat Commun. 5, (2014).</li><li>Zhu, S., Gao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+origin+of+b-defensins.">Evolutionary origin of b-defensins.</a> Dev. Comp. Immunol. 39, 79-84 (2013).</li><li>Bonmatin, J. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+1H+NMR+study+of+recombinant+insect+defensin+A+in+water:+Resonance+assignments,+secondary+structure+and+global+folding.">Two-dimensional 1H NMR study of recombinant insect defensin A in water: Resonance assignments, secondary structure and global folding.</a> J Biomol NMR. 2 (3), 235-256 (1992).</li><li>Cornet, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refined+three-dimensional+solution+structure+of+insect+defensin+A.">Refined three-dimensional solution structure of insect defensin A.</a> Structure. 3 (5), 435-448 (1995).</li><li>Murzin, A. G., Brenner, S. E., Hubbard, T., Chothia, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCOP:+a+structural+classification+of+proteins+database+for+the+investigations+of+sequences+and+structures.">SCOP: a structural classification of proteins database for the investigations of sequences and structures.</a> J Mol Biol. 247, 536-540 (1995).</li><li>Sillitoe, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CATH:+comprehensive+structural+and+functional+annotations+for+genome+sequences.">CATH: comprehensive structural and functional annotations for genome sequences.</a> Nucleic Acids Res. 43, 376-381 (2015).</li><li>Lam, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene3D:+expanding+the+utility+of+domain+assignments.">Gene3D: expanding the utility of domain assignments.</a> Nucleic Acids Res. 44, 404-409 (2016).</li><li>Tarr, D. E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+a+reference+array+for+the+CS-ab+superfamily+of+defensive+peptides.">Establishing a reference array for the CS-ab superfamily of defensive peptides.</a> BMC Res Notes. 9, 490 (2016).</li><li>Shafee, T. M. A., Robinson, A. J., van der Weerden, N., Anderson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+homology+guided+alignment+of+cysteine+rich+proteins.">Structural homology guided alignment of cysteine rich proteins.</a> SpringerPlus. 5 (27), (2016).</li><li>Altschul, S. F., Gish, W., Miller, W., Myers, E. W., Lipman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Local+Alignment+Search+Tool.">Basic Local Alignment Search Tool.</a> J Mol Biol. 215 (3), 403-410 (1990).</li><li>Duckert, P., Brunak, S., Blom, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+proprotein+convertase+cleavage+sites.">Prediction of proprotein convertase cleavage sites.</a> Protein Eng Des Sel. 17 (1), 107-112 (2004).</li><li>Petersen, T. N., Brunak, S., von Heijne, G., Nielsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SignalP+4.0:discriminating+signal+peptides+from+transmembrane+regions.">SignalP 4.0:discriminating signal peptides from transmembrane regions.</a> Nat Methods. 8, 785-786 (2011).</li><li>Kobayashi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cysteine-stabilized+a-helix:+A+common+structural+motif+of+ion-channel+blocking+neurotoxic+peptides.">The cysteine-stabilized a-helix: A common structural motif of ion-channel blocking neurotoxic peptides.</a> Biopolymers. 31, 1213-1220 (1991).</li><li>Gao, B., del Carmen Rodriguez, M., Lanz-Mendoza, H., Zhu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AdDLP,+a+bacterial+defensin-like+peptide,+exhibits+anti-Plasmodium.+activity.">AdDLP, a bacterial defensin-like peptide, exhibits anti-Plasmodium. activity.</a> Biochem Biophys Res Commun. 387, 393-398 (2009).</li><li>Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MEGA6:+Molecular+Evolutionary+Genetics+Analysis.">MEGA6: Molecular Evolutionary Genetics Analysis.</a> Mol Biol Evol. 30 (12), 2725-2729 (2013).</li><li>Edgar, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MUSCLE:+multiple+sequence+alignment+with+high+accuracy+and+high+throughput.">MUSCLE: multiple sequence alignment with high accuracy and high throughput.</a> Nucleic Acids Res. 32 (5), 1792-1797 (2004).</li><li>Ronquist, F., Huelsenbeck, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MrBayes+3:+Bayesian+phylogenetic+inference+under+mixed+models.">MrBayes 3: Bayesian phylogenetic inference under mixed models.</a> Bioinformatics. 19 (12), 1572-1574 (2003).</li><li>Altschul, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gapped+BLAST+and+PSI-BLAST:+a+new+generation+of+protein+database+search+programs.">Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.</a> Nucleic Acids Res. 25 (17), 3389-3402 (1997).</li><li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+sequence+similarity+searches+using+patterns+as+seeds.">Protein sequence similarity searches using patterns as seeds.</a> Nucleic Acids Res. 26 (17), 3986-3990 (1998).</li></ol>

The criteria for naming a protein within a group should be clear, but this is not always the case. Sequences that have the CS-&#945;&#946; fold have been studied in many labs using a variety of organisms, resulting in different systems of nomenclature, as well as varying levels of characterization. Attempting to impose a completely new nomenclature is not reasonable and would result in a great deal of confusion when consulting previous literature. A reference numbering system can be used in addition to the name of a prot...

A protein&#39;s name should reflect is characteristics and relationship to other proteins. Unfortunately, names are generally assigned at the time of discovery and, as research continues, the understanding of the larger context may change. This can lead to multiple names if a protein was independently identified by more than one lab, to changes in nomenclature or in the characteristics thought to be definitive when assigning the name, and to the name no longer sufficiently differentiating the protein from others.
Invertebrate defensins provide a good example of degeneration in nomenclature and classification. The first invertebrate defensins were reported from insects, and the name &#34;insect defensin&#34; was proposed based on the perceived homology to mammalian defensins1,2. The term defensin is still used, even though it is now clear that invertebrate and mammalian defensins do not share a common ancestor3,4. Depending on the species, an invertebrate &#34;defensin&#34; may have six or eight cysteines (that form three or four disulfide bonds) and a variety of antimicrobial activities. To complicate the situation, proteins with the same characteristics as defensins are not always called &#34;defensins,&#34; such as the recently identified cremycins from Caenorhabditis remanei5. In addition, invertebrate big defensins are more likely to be evolutionarily related to vertebrate &#946;-defensins than to other invertebrate defensins6. Despite this, researchers sometimes rely on the name &#34;defensin&#34; when determining which sequences should be included in analyses.
Structural studies revealed the similarity between insect defensins and scorpion toxins7, and the CS-&#945;&#946; fold was subsequently established as the defining structural characteristic of insect defensins8. This fold defines the scorpion toxin-like (CS-&#945;&#946;) superfamily in the Structural Classification of Proteins (SCOP) database9, which currently includes five families: insect defensins, short-chain scorpion toxins, long-chain scorpion toxins, MGD-1 (from a mollusk), and plant defensins. This superfamily is synonymous with the recently described cis-defensins4 and Superfamily 3.30.30.10 in the CATH/Gene 3D database10,11. Studies from a variety of invertebrate taxa, plants, and fungi show that the names of proteins that contain this fold are not clearly related to cysteine number or bonding pattern, antimicrobial activity, or evolutionary history12.
The lack of consistency and clear criteria make it challenging to name and classify newly-identified sequences in this superfamily. A major obstacle to comparing proteins in this superfamily is that cysteines are numbered with respect to each individual sequence (the first cysteine in each sequence is C1), with no way to account for the structural role. This means that only sequences with the same number of cysteines can be compared. There is little sequence conservation other than the cysteines forming the CS-&#945;&#946; fold, which makes alignments and phylogenetic analyses difficult. By developing a numbering system that prioritizes structural features, superfamily sequences can be more easily compared and aligned. Conserved features, as well as those defining subgroups, can be visualized quickly, and new sequences can be more easily placed into the appropriate context.
This paper uses a spreadsheet software (e.g., Excel) to generate a reference numbering system for the CS-&#945;&#946; superfamily. It shows how this clarifies comparisons between sequences and applies it to new CS-&#945;&#946; sequences identified from tardigrades. Using the CS-&#945;&#946; superfamily as an example, the protocol was written to provide guidance when using sequences of interest; however, it is not intended to be specific to this superfamily or to cysteine-rich sequences. This method will likely be most useful for groups of proteins that have been researched independently in divergent taxa and/or have little overall sequence homology, with discrete characteristics that may not be easily recognized by molecular analysis software. This method requires some a priori decisions regarding important features, so it will be of limited utility if no important features have been identified. The primary goal is to show how a simple visualization of the sequence relationships can be achieved. This can then be used to inform sequence alignment and analysis, but if alignment and analysis are the primary goals, a barcode method would be a suitable alternative that has more capacity for automation13. The current method displays the features of each peptide in a linear form, so it will not be helpful for the direct visualization of 3D structure.

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	<tbody>
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			<td height="42" style="width:216px;height:42px;">BLAST webpage</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">https://blast.ncbi.nlm.nih.gov/Blast.cgi</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">EditSeq (Lasergene suite)</td>
			<td style="width:71px;">DNASTAR</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">https://www.dnastar.com/t-allproducts.aspx</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">Excel 2013</td>
			<td style="width:71px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:216px;height:63px;">FigTree</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">&#160;http://tree.bio.ed.ac.uk/software/figtree/</td>
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			<td height="21" style="width:216px;height:21px;">MEGA</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>www.megasoftware.net</td>
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			<td height="42" style="width:216px;height:42px;">MrBayes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">http://mrbayes.sourceforge.net/</td>
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			<td height="42" style="width:216px;height:42px;">SCOP database</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">http://scop.mrc-lmb.cam.ac.uk/scop/</td>
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creating and applying a reference to facilitate the discussion and classification of proteins in a diverse group

Related proteins that have been studied in different labs using varying organisms may lack a uniform system of nomenclature and classification, making it difficult to discuss the group as a whole and to place new sequences into the appropriate context. Developing a reference that prioritizes important sequence features related to structure and/or activity can be used in addition to established names to add some coherency to a diverse group of proteins. This paper uses the cysteine-stabilized alpha-helix (CS-&#945;&#946;) superfamily as an example to show how a reference generated in&#160;spreadsheet software can clarify relationships between existing proteins in the superfamily, as well as facilitate the addition of new sequences. It also shows how the reference can help to refine sequence alignments generated in commonly used software, which impacts the validity of phylogenetic analyses. The use of a reference will likely be most helpful for protein groups that include highly divergent sequences from a broad spectrum of taxa, with features that are not adequately captured by molecular analyses.

Groups of sequences in the CS-&#945;&#946; superfamily reported in the literature are shown in Figure 4. The cysteine pairings based on the numbering for each sequence suggest five basic groups (Table 1, middle column). Group 1 has six cysteines that from three disulfide bonds and includes sequences from insects, arachnids, mollusks, nematodes, and fungi. Groups 2, 3, and 4 have 8 cysteines that form four disulfide bonds. Group 2 includes ins...

Watch this Scientific Journal Video about Creating and Applying a Reference to Facilitate the Discussion and Classification of Proteins in a Diverse Group at JoVE.com

Creating and Applying a Reference to Facilitate the Discussion and Classification of Proteins in a Diverse Group

The goal of this protocol is to develop a reference for divergent proteins in a group that lacks coherent criteria for nomenclature and classification. This reference will facilitate analyses and discussion of the group as a whole and can be used in addition to established names.

Related proteins that have been studied in different labs using varying organisms may lack a uniform system of nomenclature and classification, making it ...

The overall goal of this procedure is to use standard spreadsheet software to develop a reference for divergent proteins in a group that lacks coherent criteria for nomenclature and classification. This method can help clarify relationships between related proteins that have confusing or inconsistent nomenclature, such as the cysteine-stabilized alpha-beta superfamily of defensive peptides that includes invertebrate defenses. The main advantage of this technique is that it provides a simple visual representation of the proteins of interest.To begin, identify the defining characteristics of the protein group of interest. For example, the CS alpha-beta fold in the solution structure of insect defense and aid from phormia terraenovae defines the CS alpha-beta superfamily. This fold also includes a smaller motif called the cysteine-stabilized helix, which is identified by CXXXC upstream of a CXC.The four cysteines form two disulfide bonds. To complete the CS alpha-beta motif, a third disulfide bond is formed by an additional pair of cysteines. Ultimately it is critical to know at least some important features related to the structure or function of the protein.Without this, there is no basis for generating a reference. Now, enter these defining features into a spreadsheet. Use columns for the conserved features and to represent the spaces between these features.Keep the columns wide enough to fit numbers, and give them a consistent width. In the rows, describe the sequences. To indicate a sequence as a feature, fill in the feature box with color, using the fill function.Then, to indicate the spacing between the features, enter the number of amino acids in the box between. Now, add representative sequences that have been previously established as members of the group based on structural databases and published results. As needed, add features that are likely to define a subgroup of sequences, such as an extra cysteine.If features are missing from a given sequence, leave the box unfilled and combine it with boxes representing intervening amino acids. To do this, us the Merge and Center function. Once representative sequences have been entered, identify groups of clearly related sequences.Then, summarize the characteristics of these groups. When the number of amino acids between features varies, use a hyphen to indicate a range or slashes to indicate a few specific numbers. As needed, creatively annotate features that may be relevant or aren't common enough to include in the reference.For example, since cysteines are important in the superfamily, the presence of additional cysteines can be labeled. Sometimes, when adding newly identified sequences to the spreadsheet, the sequences of one species fall into several different groups of the superfamily, such as for tardigrades. Once completed, the rows of the spreadsheet can then be sorted to highlight variation within a species, or variation in between taxonomic groups.This demonstration uses the freely available MEGA 6 software. However, other software can be used similarly. To start a new alignment, select Edit/Build Alignment under the Align tab.Then select Create a new alignment, in the box that appears and click OK.Then select Protein. Now, select Insert Sequence from File, in the Edit menu, to import the sequences. The sequences must be in FASTA format.Background colors reflecting different amino acid types are shown by default and can be turned off with a toggle, under the Display menu. Once all the sequences are entered, click the flexing arm icon and then Align Protein to align the sequences using the muscle algorithm. If the message, Nothing selected for alignment.Select all? pops up, choose OK.Some parameters can be changed in the pop-up window, but for this demonstration, the defaults will suffice. Now, check the alignment based on the important features of the protein superfamily.The top bar shows an asterisk in any position in which the amino acid is completely conserved. The initial alignment identifies three of the four conserved cysteines. One sequence is clearly misaligned.To fix a misaligned sequence, highlight the dashes and press the Delete key without accidentally deleting amino acids. Then, move the amino acids into their proper alignment by adding spaces. After aligning the sequence manually, note that the last cysteine of the CXXXC motif is conserved throughout the alignment.Manual adjustment is often necessary to prioritize the most important features of the sequences. The spreadsheet alignment of the previously established CS alpha-beta superfamily revealed five basic patterns of bond formation. The newly identified tardigrade sequences fell into the complete spectrum of these patterns.Next, phylogenetic analyses were used to examine how this group of proteins may have evolved. However, the sequences are generally short and highly divergent. Thus, the resulting trees were poorly resolved and offered little insight.The multiple sequence alignment was optimized using the reference, but there was still poor resolution in the maximum likelihood analysis and in the Bayesian phylogenetic analysis. Most clades had only low levels of support. However, five small groups were supported in at least one of the two trees.Sequences with different cysteine numbers within a taxonomical group may be more closely related than sequences with the same pattern from different groups. After watching this video, you should have a good understanding of how to use spreadsheet software to generate a simple visualization of important protein characteristics. While attempting this procedure, it's important to remember that determining the most relevant characteristics for the group of proteins is often an iterative process, and revision of the reference will likely be necessary.New sequences can always be added to a spreadsheet, and it is easy to have multiple versions allowing for new features to be added that may be useful for classification and analysis. Although the spreadsheet alignment allows easy visualization of structural and functional features, other methods like phylogenetic analysis can be performed to provide additional insight into evolutionary relationships.

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