Interaction of hydroxylated collagen IV with the von hippel-lindau tumor suppressor
Authors: Norma Masson
Alex N Bullock
Benedikt Kessler
Alexandra Grosfeld
Matthew E Cockman
Ineke P Stolze
Peter J Ratcliffe
Christopher W Pugh
Mariola Edelmann
Journal: Journal of Biological Chemistry (2007)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: von Hippel-Lindau tumor suppressor; pVHL; hypoxia-inducible factor; HIF; ubiquitin-mediated proteasomal destruction
Affiliations: Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford OX3 7BN, United Kingdom.

Abstract:  The von Hippel-Lindau tumor suppressor (pVHL) targets hydroxylated alpha-subunits of hypoxia-inducible factor (HIF) for ubiquitin-mediated proteasomal destruction through direct interaction with the hydroxyproline binding pocket in its beta-domain. Although disruption of this process may contribute to VHL-associated tumor predisposition by up-regulation of HIF target genes, genetic and biochemical analyses support the existence of additional functions, including a role in the assembly of extracellular matrix. In an attempt to delineate these pathways, we searched for novel pVHL-binding proteins. Here we report a direct, hydroxylation-dependent interaction with alpha-chains of collagen IV. Interaction with pVHL was also observed with fibrillar collagen chains, but not the folded collagen triple helix. The interaction was suppressed by a wide range of tumor-associated mutations, including those that do not disturb the regulation of HIF, supporting a role in HIF-independent tumor suppressor functions. 

Retracted: No

PubPeer comments:
 Figure 1A. Much more similar than expected. ![file] , 
 Figure 5A. Much more similar than expected. https: imgur.com 059vPF9 , 
 Fig 1C: Not sure why there is a horizontal splice. ![file] ![file] , 
 Fig 3: More splices. ![file] ![file] , 
 Fig 5B: More splicing ![file] ![file] , 
 Thank you for your enquiries. I am going through each of these carefully both within my own laboratory and, in the case of collaborative work with any other laboratory that was involved. This will take a little time, but I will endeavour to get back with answers to your questions as soon as is possible. , 
 Figure 1a Thank you. We are not completely sure of your concern here, but assume that by stating that marks on the illustration are more similar than expected, you are concerned that these represent reproductions of an original source which has been covertly duplicated. This is not the case. We are attaching an image of the original gel and highlight the portion that was illustrated (in red box). ![image] Figure 1c You ask why there is apparently a horizontal splice towards the bottom of this gel. We attach the relevant raw data i.e. autoradiographs taken with two different exposures. The horizontal splice is not present on the originals. ![image] Essentially, we do not know how or when the apparent splice was introduced. It is not annotated in the published figure, does not interfere with, or alter, the actual data or its interpretation. Nevertheless, we apologize if this error occurred in our laboratory. The conclusion from this gel (that collagen IV, but not fibronectin can be captured from the re-immunoprecipitation of the HA IP) is not affected. All these species are clearly observed at the top of the gel and are as annotated. In this case, you have not commented on the vertical splice. For clarity, this represents juxtaposition of the two autoradiographs pertaining to the immunoprecipitation (IP) and re-IP. This was done to illustrate both at interpretable intensity, as, clearly, re-IP will only capture a proportion of the material from the IP. Figure 3 You are pointing out that the illustrations contain splices of portions of an original image. This was done to remove lanes that are not relevant to the purpose of the illustration. There was no intention to deceive, the illustration represents the actual data accurately. We have been able to find the original raw data which we attach. You will see that the excisions remove the marker lanes (and in one case an irrelevant lane). ![image] Figure 5a You are stating that marks on the illustration are much more similar than expected. We are not completely sure what you are implying, but assume you are concerned that material was covertly duplicated. Middle panel (your right hand panel). We are attaching the original gels that were illustrated, showing interaction of transfected collagen IV alpha3 u2013V5 (PK) with VHL-HA. You will notice that this original experiment also included a V5 (PK) IP that was used to verify assignment of the collagen IV alpha3 u2013V5 species in the HA IP. ![image] Left panel. We are again attaching the original gel, which shows interaction of transfected collagen 1 alpha1 with VHL-HA, which indicates that there was no duplication. ![image] However, in going over the data again, we have found an error on our part. This experiment also included a co-transfection with alpha 1 plus alpha 2. It appears that the alpha 1 plus alpha 2 HA IP lane (green *) has been illustrated in error. However, you will note that the transfected alpha 1 alone also interacts with VHL-HA (blue *). So, the result is valid but there appears to have been an error on our part for which we apologize. Figure 5b Thank you. Yes the figure was spliced. We attach the original from which it can be seen that controls and comparators (BSA and fibronectin) were correctly taken from the same gel. ![image] The point of this figure is to illustrate that collagen IV binds more strongly after denaturation. As we discuss at the start of discussion this (i.e. unfolded collagen chain existence) might only be predicted to occur physiologically during assembly. Though it was not the main part of the paper (since the primary affinity capture with VHL was collagen IV) u2013 we also tested whether fibrillary collagens would interact with VHL when denatured, and the figure shows that this is the case. You will see that we have not illustrated this for collagen V where the denaturation-dependent increase in binding is clear but a weaker interaction with apparently native material is observed. It is difficult to know whether all the collagen in the supplied preparation was indeed folded and hence how to interpret the existence of some interaction with the supposedly entirely native material. You may argue that this data should have been included though it does not affect, and is peripheral to the main conclusion of the paper - and had the potential to confuse (since the supposedly native collagen V may not have been fully native). Hence, we did not do so. The main findings on the interaction of collagen IV with VHL, that this interaction is direct, that the interaction is dependent on prolyl hydroxylation and that it is perturbed by tumour associated mutations in VHL have all been reproduced by others (Kurban et al. Oncogene 27 10004-1012, 2008). We believe that the above answers your questions as fully and accurately as we are able, and that the main conclusions of the paper are secure. However, in the interests of full clarity we will report this exchange to the Journal in case they wish to consider further. Peter Ratcliffe and Norma Masson , 
 #7  Figure 5a You are stating that marks on the illustration are much more similar than expected. We are not completely sure what you are implying, but assume you are concerned that material was covertly duplicated. Figure 5A. Much more similar than expected. ![file] , 
 #8 I agree that these lanes appear to unexpectedly have similarly placed background features. *** Inverse, contrast enhanced ![file] *** Forensically ![file] , 
 Figure 5A. I see image tool and splice ![file] ![file] , 
 5A. Splice and more similar than expected ![file] ![file] , 
 Thank you for clarifying your exact issues with Figure 5A. Middle panel (your right hand panel): If we now understand correctly you are saying that the published figure has a horizontal splice, that there is a duplication of a control lane and that the RHS lanes have been moved down with respect to the LHS lanes. We accept this and apologise that these manipulations must have occurred during construction of the figure. The raw data (attached earlier) clearly shows the integrity of the scientific result and so we are disappointed to find these (unnecessary) manipulations in our published figure. Left panel: The raw data (attached earlier) clearly shows the integrity of the scientific result and so again we are disappointed to find these unnecessary changes in the published figure. We apologize for these errors. We contacted JBC yesterday with regards to the Pubpeer comments. We will update them with this further exchange and follow their advice. Norma Masson and Peter Ratcliffe , 
 Thank you for clarifying your exact issues with Figure 5A. Middle panel (your right hand panel): If we now understand correctly you are saying that the published figure has a horizontal splice, that there is a duplication of a control lane and that the RHS lanes have been moved down with respect to the LHS lanes. We accept this and apologise that these manipulations must have occurred during construction of the figure. The raw data (attached earlier) clearly shows the integrity of the scientific result and so we are disappointed to find these (unnecessary) manipulations in our published figure. Left panel: The raw data (attached earlier) clearly shows the integrity of the scientific result and so again we are disappointed to find these unnecessary changes in the published figure. We apologize for these errors. We contacted JBC yesterday with regards to the Pubpeer comments. We will update them with this further exchange and follow their advice. Norma Masson , 
 Not sure what might contribute to this artifact also in Figure 5a ![file] ![file] , 
 Thank you for your further comment. The gel was cut in order to assemble a figure portraying the most relevant data to the argument being made. We agree that it would be better practice to annotate any cuts, or otherwise make them completely clear, and to submit copies of the full gels to the journal. But that is in hindsight. At the time, cutting gels to remove extraneous information was common practice in this laboratory (as it was in many others). There was no intention of deceit on our part and we did not consider that such practice would be used to deceive (in any laboratory). This is not a defence of these practices, but an explanation. As you will be aware any illustration of data is just that i.e. a representative selection of relevant data that exemplifies the experimental evidence. As such, whatever practice is used, it is dependent on the integrity of the investigator. In the case of this panel we have not so far been able to locate the original autoradiograph. From our recollection, laboratory records, and other data, we are confident that the result portrayed is correct i.e. that the non-collagenous domain of the collagen chain interacts much less strongly with VHL than the collagenous domain, in accordance with their relative levels of prolyl hydroxylation. However, to remove any possible doubt that your enquiries might have raised in the minds of others, we will repeat this experiment and make the result available. , 
 Please find attached a repetition of the experiment originally illustrated in Figure 5A right hand panel. Fig 5A seeks to compare the interactions that occur between pVHL and full-length collagen chains with those of an isolated non-collagenous domain (from collagen IValpha3). For this reason, both interactions with the full-length chain (collagen IValpha3) and the isolated NC1 domain were re-assessed in parallel and a representative dataset is provided. You will see that the ratio of captured species to input (lanes 7 lane 3) in each panel is very much higher for the full-length collagen chain than with the isolated non-collagenous domain (we estimate 50x from this and other experiments). This corroborates the result in the original illustration. Norma Masson and Peter Ratcliffe ![file] ,