Neurology -- Correspondence for Cummins et al., 60 (2) 224-229

Correspondence published:

Reply to Letter to the Editor: T R Cummins, M Reganathan, P K Stys, R I Herzog, R Horn, S D Dib-Hajj, and S G Waxman (11 April 2003)

The pentapeptide QYNAD does not block voltage-gated sodium channels: Heinrich Brinkmeier, Frank Weber, Peter Aulkemeyer, Kurt H Wollinsky and Reinhardt Rudel (11 April 2003)

Reply to Letter to the Editor 11 April 2003

T R Cummins
Yale University,
M Reganathan, P K Stys, R I Herzog, R Horn, S D Dib-Hajj, and S G Waxman
Send Correspondence to journal:
Re: Reply to Letter to the Editor

theodore.cummins{at}yale.edu T R Cummins, et al.

In our paper [1] we suggested that the conclusion of Brinkmeier et al. [2] that QYNAD acts as a sodium channel-blocking factor be viewed with caution. This position was based on results of experiments carried out in three different laboratories, all of which failed to provide any evidence for modulation or block of sodium channels, by QYNAD. These experiments used QYNAD synthesized in 10 different batches, including three separate batches from the facility that prepared QYNAD for the original report by the Rüdel group. [2] The fact that Brinkmeier et al. now report that they also have inactive batches of QYNAD for reasons that they can not identify heightens the concern about the identity of the blocking factor in their previous reports. [2, 3] The potential conversion of the N-terminal glutamine into pyroglutamic acid is unlikely to account for the total lack of activity in our experiments. QYNAD was always dissolved immediately before use in our experiments, and in a tandem mass spectrometric (MSMS) analysis of a dissolved sample that did not show modulatory and/or blocking activity, unmodified QYNAD was readily detected but pyQYNAD was not. Furthermore, we have tested concentrations of QYNAD that are 10 fold higher than the saturation concentration reported by Brinkmeier et al. [2] and should presumably have been effective if only a fraction of the peptide maintained native chemistry. Additionally, three QYNAD batches modified by N-terminal acylation with an acetyl group (which prevents pyQYNAD formation) were also ineffective in blocking and/or modulating sodium currents. These experiments were undertaken based on the assertion by Brinkmeier et al in their original report that "acylation of the N- terminal amino group caused only a minor reduction of its blocking activity." [2] It is surprising that Brinkmeier et al. now state that N- terminal acylation of QYNAD inactivates the peptide, and this change in their position raises additional concerns regarding the interpretation of their original experimental data. The fact that 10 different batches of QYNAD prepared by four different facilities (including those used by Brinkmeier et al. [2]) and tested by several labs were inactive in the sodium channel assays, [1] coupled with the unexplained change in position by Brinkmeier et al. regarding the effect of acylation of QYNAD and their current report of inactive batches of QYNAD peptide, [4] raises very significant concerns about the suggestion that QYNAD peptide is a sodium channel blocker or modulator.
References:
1. Cummins TR, Renganathan M, Stys PK, Herzog RI, Scarfo K, Horn R, Dib-Hajj SD, Waxman SG. The pentapeptide QYNAD does not block voltage- gated sodium channels. Neurology 2003;60:224-229.
2. Brinkmeier H, Aulkemeyer P, Wollinsky KH, Rudel R. An endogenous pentapetide acting as a sodium channel blocker in inflammatory autoimmune disorders of the central nervous system. Nature Medicine 2000;6:808-811.
3. Weber F, Rudel R, Aulkemeyer P, Brinkmeier H. The endogenous pentapeptide QYNAD induces acute conduction block in the isolated rat sciatic nerve. Neurosci Lett 2002;317:33-36.
4. Brinkmeier H, Weber F, Aulkemeyer P, Wollinsky KH, Rudel R. Letter to editor.

The pentapeptide QYNAD does not block voltage-gated sodium channels 11 April 2003

Heinrich Brinkmeier
Ulm and Greifswald Germany,
Frank Weber, Peter Aulkemeyer, Kurt H Wollinsky and Reinhardt Rudel
Send Correspondence to journal:
Re: The pentapeptide QYNAD does not block voltage-gated sodium channels

heinrich.brinkmeier{at}uni-greifswald.de Heinrich Brinkmeier, et al.

We read with interest the paper by Cummins et al. [1], which addresses our paper reporting the isolation and sequencing of the pentapeptide QYNAD from the CSF of MS and GBS patients. In it, we claim that the isolated fraction as well as the synthesized peptide blocks neuronal sodium channels. [2] Cummins et al., testing several batches of QYNAD synthesized by various producers could not confirm this. Several possibilities may cause the discrepancy.
For our report, we had used three different batches of QYNAD synthesized by Interactiva, Ulm, which all showed about the same blocking efficacy. Beginning with spring 2001, we noted that most of the newly supplied batches were inactive for reasons that we could not identify. Active batches had been consistently effective in all tested models, NH15- CA2 neuroblastoma cells heterologously expressed rat Nav 1.2 and isolated rat sciatic nerve;[3] inactive batches had no effect in any model. Also, QYNAD ordered from EMC microcollections (Tübingen, Germany) was inactive. Meanwhile a group working completely independent from us and using QYNAD synthesized from a university lab, reproduced our results with isolated rat thalamic neurons4. We tested one of their batches on Nav 1.2 and found it as active as our samples. As all these supplies had 95% purity guaranteed, the contrasts in activity are not easily explained. Different methods of synthesis, purification or lyophilization could account for them. We also had acetylated, amidated or mutant variants of QYNAD synthesized. All were inactive, reassuring us that Na+ channel blockade was QYNAD-specific or at least QYNAD-related, certainly not unspecific. One known effect can contribute to reduced QYNAD activity. Particularly when above room temperature, the N-terminal glutamine (Q) gives off an NH3 residue to form the pyroglutamic acid derivative, pyQYNAD. We detect it regularly by its reduced mass (592.2 vs. 609.2 Da). In CSF, the reaction seems to be slower, perhaps due to the presence of a stabilizing co-factor. The earlier used MALDI-TOF mass spectrometry is not quantitative, and therefore does not give information about the QYNAD/pyQYNAD ratio during experimentation. We have now installed the more sensitive ion trap technique for mass detection and quantification and find QYNAD and pyQYNAD in CSF samples of all assessed MS and of GBS patients, but not of controls.
Working on questions such as whether QYNAD is degraded during experimentation or requires a co-factor for its blocking effect, we still believe in its biological importance in demyelinating diseases.
References:
1. Cummins TR, Renganathan M, Stys PK, Herzog RI, Scarfo K, Horn R, Dib-Hajj SD,Waxman SG. The pentapeptide QYNAD does not block voltage-gated sodium channels. Neurology 2003;60:224-229.
2. Brinkmeier H, Aulkemeyer P, Wollinsky KH, Rüdel R. An endogenous pentapeptide acting as a sodium channel blocker in inflammatory autoimmune disorders of the central nervous system. Nat Med 2000;6:808-811.
3. Weber F, Rüdel R, Aulkemeyer P, Brinkmeier H. The endogenous pentapeptide QYNAD induces acute conduction block in the isolated rat sciatic nerve. Neurosci Lett 2002;317: 33-36.
4. Wiendl H, Meuth S, Duyar H, Elbs M, Landgraf P, Weller M, Weissert R, Budde T.Modulation of neuronal activity and MOG-induced EAE by the endogenous pentapeptide, QYNAD, a putative mediator of neurological dysfunction in human demyelinating diseases. Mult Scler 2002;8:S54.