Myriapod haemocyanin: the first three-dimensional reconstruction of Scolopendra subspinipes and preliminary structural analysis of S. viridicornis.

Haemocyanins (Hcs) are copper-containing, respiratory proteins that occur in the haemolymph of many arthropod species. Here, we report the presence of Hcs in the chilopode Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The analysis of transcriptome of S. subspinipes subpinipes reveals the presence of two distinct subunits of Hc, where the signal peptide is present, and six of prophenoloxidase (PPO), where the signal peptide is absent, in the 75 kDa range. Size exclusion chromatography profiles indicate different quaternary organization for Hc of both species, which was corroborated by TEM analysis: S. viridicornis Hc is a 6 × 6-mer and S. subspinipes Hc is a 3 × 6-mer, which resembles the half-structure of the 6 × 6-mer but also includes the presence of phenoloxidases, since the 1 × 6-mer quaternary organization is commonly associated with hexamers of PPO. Studies with Chelicerata showed that PPO activity are exclusively associated with the Hcs. This study indicates that Scolopendra may have different proteins playing oxygen transport (Hc) and PO function, both following the hexameric oligomerization observed in Hcs.

Hcs are grouped together with phenoloxidases (POs), having a very similar architecture of the respective actives sites. POs are part of the innate immune system, and are for example involved in wound healing and sclerotization. In fact, chelicerata do not seem to possess POs, and the respective functions is thought to be performed by Hcs, activated e.g. by proteolytic enzymes. The quaternary structure of Hcs of arthropoda comprises multiples of hexamers. Typically, in crustacean 1x6 or 2x6 variants are found, in chelicerates additionally 4x6 or 8x6mers. Characteristic for myriapoda seem to be formation of 3x6 or 6x6 mers, but characterization of the quaternary structure was performed so far only in three cases. Typically, larger quaternary assemblies require a larger number of different subunit types, and go along with a lower affinity, and higher cooperativity. Arthropod POs are usually found as hexamers in the hemolymph, typically as pro-enzymes. Compared to HCs the amount of POs is very small, while Hc concentration can be as high as 100 mg/ml in certain species. In the present manuscript, Hcs and POs from another chilopoda, namely two species of the order Scolopendromorpha (Scolopendra suspinipes and Sclolopendra viridicornis) are described. To this end, the sequences were determined, the transcription level addressed and, for the hemocyanin, the protein isolated and the quaternary structure determined by 3D-EM reconstruction. Overall, the paper seems to be sound. However, the presentation needs to be improved, both with respect to quality of the figures and use of English language (some passages are practically uncomprehensible). With respect to this, I only pointed to a few passages. The complete manuscript has to be thoroughly revised in terms of writing.
Specific comments: Tab. 1 in S1 undecipherable Page 5: structure of PO: not all POs are hexameric, but the one from crustacean are. Page 5: The text jumps quite a bit between the different phylogenetic levels. Please be more correct, not every reader is familiar with the myriapod phylogeny. Fig.1: species undecipherable, please reconsider presentation. p.9: the discussion about the role of tryptophan needs to be revised, too general and not really clear, what the important factor with respect to Hc/Po function actually is. Fig.3: "Hemocytes and plasma from both species had protein dispersed…". I don't see anything in case of SviH. I also cannot see six bands in SsuH. Overall, it is difficult to relate the fig. to the text. Please reconsider. Possibly the gels can be omitted. p.11: The sententences "..activity was observed only in hemocytes after activation …by trypsin" and "We observed the auto-activation of the hemocytes…" seem to be contradictory in their meaning. Please clarify. Why is the activity lower in presence of SDS? Usually POs can be activated by SDS…. PO presents a much higher fraction of protein in hemocytes than in plasma, therefore a comparison based of the overall protein concentration is difficult, and therefore one cannot exclude that POs play a role in plasma also. Also reconsider the respective sentences in the discussion (p. 21. Last paragraph). Fig. 4, legend: what is meant by "dopamine like substrate"? p.12: the number of particles on the EM grids at the different pH values etc could be better acknowledged if presented as bar chart. Page 14: SEC profiles: axis title missing. Please specify the SEC column in more detail (supplier, complete name We are writing to inform you that the Editor has reached a decision on your manuscript RSOB-19-0258 entitled "Myriapod Hemocyanin: The First 3D Reconstruction of Scolopendra subspinipes and Preliminary Structural Analysis of S. viridicornis", submitted to Open Biology.
As you will see from the reviewer's comments below, there are a number of criticisms that prevent us from accepting your manuscript at this stage. The reviewers suggest, however, that a revised version could be acceptable, if you are able to address their concerns. If you think that you can deal satisfactorily with the reviewer's suggestions, we would be pleased to consider a revised manuscript.
The revision will be re-reviewed, where possible, by the original referees. As such, please submit the revised version of your manuscript within four weeks. If you do not think you will be able to meet this date please let us know immediately.
To revise your manuscript, log into https://mc.manuscriptcentral.com/rsob and enter your Author Centre, where you will find your manuscript title listed under "Manuscripts with Decisions." Under "Actions," click on "Create a Revision." Your manuscript number has been appended to denote a revision.
You will be unable to make your revisions on the originally submitted version of the manuscript. Instead, please revise your manuscript and upload a new version through your Author Centre.
When submitting your revised manuscript, please respond to the comments made by the referee(s) and upload a file "Response to Referees" in "Section 6 -File Upload". You can use this to document any changes you make to the original manuscript. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response to the referee(s).
Please see our detailed instructions for revision requirements https://royalsociety.org/journals/authors/author-guidelines/ Once again, thank you for submitting your manuscript to Open Biology, we look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch.
Sincerely, The Open Biology Team mailto: openbiology@royalsociety.org Reviewer's Comments to Author(s): Referee: Comments to the Author(s) Hemocyanins (Hcs) are oxygen transport proteins exclusively found in certain invertebrates, namely molluscs and arthropods. Within the phylum of arthropods, Hcs are mainly found in chelicerates and crustacean, but also in some hexapoda and myriapoda. Within the latter subphylum, Hcs have been found in diplopoda, and in one case in chilopoda, in the order scutigeropmorpha. Hcs are grouped together with phenoloxidases (POs), having a very similar architecture of the respective actives sites. POs are part of the innate immune system, and are for example involved in wound healing and sclerotization. In fact, chelicerata do not seem to possess POs, and the respective functions is thought to be performed by Hcs, activated e.g. by proteolytic enzymes. The quaternary structure of Hcs of arthropoda comprises multiples of hexamers. Typically, in crustacean 1x6 or 2x6 variants are found, in chelicerates additionally 4x6 or 8x6mers. Characteristic for myriapoda seem to be formation of 3x6 or 6x6 mers, but characterization of the quaternary structure was performed so far only in three cases. Typically, larger quaternary assemblies require a larger number of different subunit types, and go along with a lower affinity, and higher cooperativity. Arthropod POs are usually found as hexamers in the hemolymph, typically as pro-enzymes. Compared to HCs the amount of POs is very small, while Hc concentration can be as high as 100 mg/ml in certain species. In the present manuscript, Hcs and POs from another chilopoda, namely two species of the order Scolopendromorpha (Scolopendra suspinipes and Sclolopendra viridicornis) are described. To this end, the sequences were determined, the transcription level addressed and, for the hemocyanin, the protein isolated and the quaternary structure determined by 3D-EM reconstruction. Overall, the paper seems to be sound. However, the presentation needs to be improved, both with respect to quality of the figures and use of English language (some passages are practically uncomprehensible). With respect to this, I only pointed to a few passages. The complete manuscript has to be thoroughly revised in terms of writing.
Specific comments: Tab. 1 in S1 undecipherable Page 5: structure of PO: not all POs are hexameric, but the one from crustacean are. Page 5: The text jumps quite a bit between the different phylogenetic levels. Please be more correct, not every reader is familiar with the myriapod phylogeny. Fig.1: species undecipherable, please reconsider presentation. p.9: the discussion about the role of tryptophan needs to be revised, too general and not really clear, what the important factor with respect to Hc/Po function actually is. Fig.3: "Hemocytes and plasma from both species had protein dispersed…". I don't see anything in case of SviH. I also cannot see six bands in SsuH. Overall, it is difficult to relate the fig. to the text. Please reconsider. Possibly the gels can be omitted. p.11: The sententences "..activity was observed only in hemocytes after activation …by trypsin" and "We observed the auto-activation of the hemocytes…" seem to be contradictory in their meaning. Please clarify. Why is the activity lower in presence of SDS? Usually POs can be activated by SDS….
PO presents a much higher fraction of protein in hemocytes than in plasma, therefore a comparison based of the overall protein concentration is difficult, and therefore one cannot exclude that POs play a role in plasma also. Also reconsider the respective sentences in the discussion (p. 21. Last paragraph). Fig. 4

Comments to the Author
The authors addressed all of my concerns appropriately. There is only one aspect I suggest to change: if the 2D-Gel are not shown, they should not be discussed, and the corresponding part in the methods-part should be deleted.
My problem was, that I cannot see what the authors describe, like six bands in case of Ssuh. This problem does not disappear by not showing the gels. So I suggest to completely deleted all parts refering to the gels. That does not change the story.

02-Mar-2020
Dear Dr da Silva Junior, We are pleased to inform you that your manuscript RSOB-19-0258.R1 entitled "Myriapod Hemocyanin: The First 3D Reconstruction of Scolopendra subspinipes and Preliminary Structural Analysis of S. viridicornis" has been accepted by the Editor for publication in Open Biology. The reviewer(s) have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, we invite you to respond to the reviewer(s)' comments and revise your manuscript.
Please submit the revised version of your manuscript within 7 days. If you do not think you will be able to meet this date please let us know immediately and we can extend this deadline for you.
To revise your manuscript, log into https://mc.manuscriptcentral.com/rsob and enter your Author Centre, where you will find your manuscript title listed under "Manuscripts with Decisions." Under "Actions," click on "Create a Revision." Your manuscript number has been appended to denote a revision.
You will be unable to make your revisions on the originally submitted version of the manuscript. Instead, please revise your manuscript and upload a new version through your Author Centre.
When submitting your revised manuscript, you will be able to respond to the comments made by the referee(s) and upload a file "Response to Referees" in "Section 6 -File Upload". You can use this to document any changes you make to the original manuscript. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response to the referee(s).
Before uploading your revised files please make sure that you have: 1) A text file of the manuscript (doc, txt, rtf or tex), including the references, tables (including captions) and figure captions. Please remove any tracked changes from the text before submission. PDF files are not an accepted format for the "Main Document".
2) A separate electronic file of each figure (tiff, EPS or print-quality PDF preferred). The format should be produced directly from original creation package, or original software format. Please note that PowerPoint files are not accepted.
3) Electronic supplementary material: this should be contained in a separate file from the main text and meet our ESM criteria (see http://royalsocietypublishing.org/instructions-authors#question5). All supplementary materials accompanying an accepted article will be treated as in their final form. They will be published alongside the paper on the journal website and posted on the online figshare repository. Files on figshare will be made available approximately one week before the accompanying article so that the supplementary material can be attributed a unique DOI.
Online supplementary material will also carry the title and description provided during submission, so please ensure these are accurate and informative. Note that the Royal Society will not edit or typeset supplementary material and it will be hosted as provided. Please ensure that the supplementary material includes the paper details (authors, title, journal name, article DOI). Your article DOI will be 10.1098/rsob.2016[last 4 digits of e.g. 10.1098/rsob.20160049]. 4) A media summary: a short non-technical summary (up to 100 words) of the key findings/importance of your manuscript. Please try to write in simple English, avoid jargon, explain the importance of the topic, outline the main implications and describe why this topic is newsworthy.

Images
We require suitable relevant images to appear alongside published articles. Do you have an image we could use? Images should have a resolution of at least 300 dpi, if possible.

Data-Sharing
It is a condition of publication that data supporting your paper are made available. Data should be made available either in the electronic supplementary material or through an appropriate repository. Details of how to access data should be included in your paper. Please see http://royalsocietypublishing.org/site/authors/policy.xhtml#question6 for more details.
Data accessibility section To ensure archived data are available to readers, authors should include a 'data accessibility' section immediately after the acknowledgements section. This should list the database and accession number for all data from the article that has been made publicly available, for instance: We are pleased to inform you that your manuscript entitled "Myriapod Hemocyanin: The First 3D Reconstruction of Scolopendra subspinipes and Preliminary Structural Analysis of S. viridicornis" has been accepted by the Editor for publication in Open Biology.
You can expect to receive a proof of your article from our Production office in due course, please check your spam filter if you do not receive it within the next 10 working days. Please let us know if you are likely to be away from e-mail contact during this time.
Article processing charge Please note that the article processing charge is immediately payable. A separate email will be sent out shortly to confirm the charge due. The preferred payment method is by credit card; however, other payment options are available.
Thank you for your fine contribution. On behalf of the Editors of Open Biology, we look forward to your continued contributions to the journal.

Reviewer's Comments to Author(s):
Referee: Comments to the Author(s) Hemocyanins (Hcs) are oxygen transport proteins exclusively found in certain invertebrates, namely molluscs and arthropods. Within the phylum of arthropods, Hcs are mainly found in chelicerates and crustacean, but also in some hexapoda and myriapoda. Within the latter subphylum, Hcs have been found in diplopoda, and in one case in chilopoda, in the order scutigeropmorpha. Hcs are grouped together with phenoloxidases (POs), having a very similar architecture of the respective actives sites. POs are part of the innate immune system, and are for example involved in wound healing and sclerotization. In fact, chelicerata do not seem to possess POs, and the respective functions is thought to be performed by Hcs, activated e.g. by proteolytic enzymes. The quaternary structure of Hcs of arthropoda comprises multiples of hexamers. Typically, in crustacean 1x6 or 2x6 variants are found, in chelicerates additionally 4x6 or 8x6mers. Characteristic for myriapoda seem to be formation of 3x6 or 6x6 mers, but characterization of the quaternary structure was performed so far only in three cases. Typically, larger quaternary assemblies require a larger number of different subunit types, and go along with a lower affinity, and higher cooperativity. Arthropod POs are usually found as hexamers in the hemolymph, typically as pro-enzymes. Compared to HCs the amount of POs is very small, while Hc concentration can be as high as 100 mg/ml in certain species. In the present manuscript, Hcs and POs from another chilopoda, namely two species of the order Scolopendromorpha (Scolopendra suspinipes and Sclolopendra viridicornis) are described. To this end, the sequences were determined, the transcription level addressed and, for the hemocyanin, the protein isolated and the quaternary structure determined by 3D-EM reconstruction. Overall, the paper seems to be sound. However, the presentation needs to be improved, both with respect to quality of the figures and use of English language (some passages are practically uncomprehensible). With respect to this, I only pointed to a few passages. The complete manuscript has to be thoroughly revised in terms of writing.
Specific comments: Tab. 1 in S1 undecipherable Answer: I Changed the figure  sequences of hemocyanin with PO activity (EcaHcA-G); in contrast, our inspection of the sequence/structure of hemocyanin with only oxygen transport activity showed a valine in the crustacean protein (1HCY) and tyrosine in the myriapods ones (Fig -02; S1Figure) instead of the tryptophan [34,64] .
A conserved Phe (F) residue called "place holder" occurs in the active site pocket before activation and is thought to block access to the substrate is present in all type 3 copper protein.
When PPO is activated, this place holder must be removed from the active site pocket. When the blocking residue (F 84 ) in DmPPO3 (Drosophila melanogaster) was mutated into tryptophan (W), which has a hydrophobic side chain, DmPPO3(W 84 ) activity significantly decreased after being activated by ethanol [65].
Tryptophan contains a non-carbon atom (nitrogen) in the aromatic ring, making this residue more reactive than phenylalanine although less so than than tyrosine. Tryptophan can play a role in binding to non-protein atoms while the tyrosine side chain, being partially hydrophobic, prefers to be a buried in the hydrophobic core. Also, the aromatic tyrosine side chain can be involved in π-π stacking interactions with other aromatic side chains. The valine side chain is small but completely aliphatic and hydrophobic and hence non-reactive, and is thus rarely directly involved in protein functions like catalysis, although it can play a role in substrate recognition. The hydrophobic aromatic amino acid residues can sometimes substitute for aliphatic residues of a similar size, for example phenylalanine for leucine, but not the large tryptophan for the small valine. In particular, hydrophobic amino acid residues can be involved in binding/recognition of hydrophobic ligands such as lipids [66].
Although the di-copper active sites of all of the above-mentioned type 3 copper proteins could be well superimposed, interesting differences have been observed, mainly at the CuA site [33]. In particular, tyrosine-switched tryptophan may have made the region less hydrophobic by preventing the entry of substrate, which may explain the probable loss of PO activity from the two hemocyanin proteins identified in Scolopendra subspinipes subspinipes (Error! Reference source not found.). p.11: The sententences "..activity was observed only in hemocytes after activation …by trypsin" and "We observed the auto-activation of the hemocytes…" seem to be contradictory in their meaning. Please clarify. Answer: I changed this phrase In this study, we used a concentration of 20 µg/mL from lysate of hemocytes and plasma for both species to verify the o-diphenoloxidase incubating with dopamine. The activation of prophenoloxidase to phenoloxidase by trypsin (square gray) and the activity of the lysate of hemocytes with buffer (triangle blue) occurred for both species ( Figure 1A, B), suggesting that serine protease, the enzyme responsible for this activity, was also present in the hemocytes. Previous studies showed an activation of phenoloxidase activity for myriapods by zymosan and chymotrypsin [76], corroborating our results.
Why is the activity lower in presence of SDS? Usually POs can be activated by SDS…. PO presents a much higher fraction of protein in hemocytes than in plasma, therefore a comparison based of the overall protein concentration is difficult, and therefore one cannot exclude that POs play a role in plasma also. Also reconsider the respective sentences in the discussion (p. 21. Last paragraph). Answer: Previous studies showed an activation of phenoloxidase activity for myriapods by zymosan and chymotrypsin (Xylander WER. 1992 Immune Defense Reactions of Myriapoda -A Brief Presentation of Recent Results. 8th Int. Congr. Myriapodology), corroborating our results. Probably the phenoloxidase of plasma occurs after the hemocytes degranulation. In our experiments, we took care to avoid degranulation that is responsible to liberate the granules with PO in plasma. Thus, in this case, we can conclude that PO activity is only in hemocytes lysates. The mechanism of activation of PO in arthropods are still in study and not clean.     Observations of the mantle zone indicated a lack of any contact between two subunits from each hexamer and any other hexamerand indicated the remaining two subunits to be attached to their counterpart in the adjacent hexamer, together forming the three peripheral bridges. In the 6 x 6-mer assembly of S. coleoptrata hemocyanin, this type of bridge has been designated as a 3↔4 interface [54,57]. This interface in Ssu1 formed from residues 402HYKLTYPA409, 633TH634 and in Ssu 2 from 361VEQLTWPD368, 587TH588.
In the S. coleoptrata 6 x 6-mer structure, the two 3 x 6-mer half structures have been previously noted to be connected in the mantle by a type of inter-hexamer bridge that has been termed a 3↔3 interface [54]. We observed the presence of histidine (147NRGEHYDRIPi156, 442TKH444) in the ScoHcC subunit; histidine is an important amino acid able to participate in π-π stacking interactions and it was responsible for stabilizing the structure. Our inspection of the Ssu1 sequence at 141IRGNQ-NPVVNL154 and 432TRQ434 showed a gap in this region and the absence of an amino acid residue able to perform the same interaction. And our inspection of Ssu2 at 106QKALRNDKDIVVD118 and 401TGP403 showed an arginine in the place of the histidine; the arginine here may support allostery rather than blocking potential dimerization of 3 x 6-mers [54].
The second type of bridge connecting the 3 x 6-mers in the core of S. coleoptrata is termed the 1A↔1E interface, with this interface established by interactions of basic residues (KAKK) in the β3D → β3E loop with acidic residues (ED) at the end of helix α3.5. Comparable charges (ENKK and PSD) were observed in this region of Ssu2 (Fig. S2).
The three central inter-hexamer bridges of the 3 x 6-mer have been observed to be formed by the core subunit Ssu2, and this type of contact has been termed a 1↔2 interface [54]. Each of the three bridges was formed by a pair of such interfaces together being made up of a large number of residues (Error! Reference source not found. poor score and red a good score, in Error! Reference source not found.D-F. This pattern demonstrated the potential of an Ssu2 chain to interact with another Ssu2 chain in several different ways, or that Ssu2 could be interacting with more than one chain simultaneously. Assuming the last hypothesis, the Ssu2 chain would be localized in the middle (core zone) of the 3 x 6 hexamer and interacting with other Ssu2 chains inside the hexamer (1↔2 interface) and hence could provide inter-hexamer interactions.  Based on symmetry considerations previously determined for the S. coleoptrata 6 x 6-mer, and applied to the 3 x 6-mer found in S. subspinipes subspinipes, we suggested that the constituent p.22, second last paragraph: typically the concentration of POs in the hemolymph is much lower than Hc. Thus, it seems unlikely that the observed 1x6mers are POs. Please rephrase the corresponding sentence in the abstract. Answer: I changed the phrase. While we found a relatively small amount of the 3 x 6-mer hemocyanin form in Scolopendra subspinipes subspinipes and found the 6 x 6-mer form in Scolopendra viridicornis, phenoloxidases were also found to be present in the hemolymph. Note in this regard that the 1 x 6-mer quaternary organization has also been shown for hexamers of PPO [34]. More studies of hemocyanin of Scolopendra viridicornis will be necessary to explain the presence of 6 x 6-mer hemocyanin.