Question And Answer Session

DR. BASLOW: Now we're open to questions. I have one question, if I may. Would you say that this acetate is necessary for the buildup of myelin?

DR. LEDEEN: Oh, no. I should make that point clear. This is a pathway that is not the primary source of myelin. We see it more as a later addition to the repertoire of myelin synthesis, perhaps maintenance and turnover of myelin.

After the CNS myelin has been synthesized, it would be a tremendous job for one little oligodendrocyte to maintain these 50 internodes or so that it has put out. And we think in that case, there is the need for trophic support from the axon.

So we feel it's more of a later phenomenon, although we don't preclude the possibility that it might also have a role earlier on but not the sole or even primary role.

DR. BASLOW: Thank you.

PARTICIPANT: As a clinician, I've never been able to satisfy myself of the answer to what that rate of myelin turnover is. Do you have a sense of the rate of myelination?

DR. LEDEEN: Oh, my. There's a long history of studies on myelin turnover rates. And there is no clear picture except we now know that the early idea that myelin does not turn over, that it is an inert membrane, is not true, that both the proteins and the lipids do turn over at variable rates. Some of the lipids turn over more rapidly than others. And one that does not turn over quite so rapidly is the one I mentioned, the ethanolamine phosphoglyceride, which is made up largely of plasmalogen form, that lipid. And that has a longer half-life. And that is the one that seems to be labeled by NAA more than the others that are turning over more rapidly.

So I suspect that's going to be part of the story that will have to be clarified as time goes on, what is the relation between this pathway and turnover, as opposed to other pathways that lead to myelin synthesis.

DR. NAMBOODIRI: I have one question regarding that high molecular weight form of your enzyme. The ASPA you find associated with myelin, you find that the molecular weight is around the 53-54?

DR. LEDEEN: Or higher, yes.

DR. NAMBOODIRI: What type of antibody did you use? Was it a peptide-based antibody or was it an antibody against the protein?

DR. LEDEEN: Well, I got the antibody from Reuben Matalon. So he can probably answer that.

SESSION CO-CHAIR MATALON: This is an antibody that we prepared from purified protein. Actually, the antibody was made with the 313 amino acids, about 36 kilodalton protein, just what we used to generate the antibody, this class of antibody. We had in the past a different antibody. And we had similar finding to what Dr. Ledeen had. In our first publication on aspartoacylase, we said that the molecular weight was about 56 kilodaltons.

When we isolated the gene and we found that it codes for 313 amino acids, we went back and tried to find out what was the cause for that big protein. And we found out that when we changed the isolation technique, we got a smaller protein. We decreased the salt concentration in the isolation prep. And we got about 36 kilodalton.

DR. KOLODNY: Is it possible, then, that there are really two different proteins, one in the cytosol and the 56, the larger protein and because aspartoacylase is an esterase and has structure that is similar to other esterases? So could the antibody, then, be reacting to more than one esterase?

SESSION CO-CHAIR MATALON: This is a polyclonal antibody. So it can have various epitopes.

So I can't tell you. It detected the 56 kilodalton in your case and also the 36 kilodalton.

DR. MADHAVARAO: It is possible that the 56 kilodalton is only in myelin, and is not the cytosoic isoform. So unless the cytosolic isoform is also purified, which has no contamination from the myelin form, it is not possible; you should see some residual activity with the cytosolic form.

SESSION CO-CHAIR MATALON: Okay. Our isolation of aspartoacylase, we used two types of preparations. One is human skin fibroblasts, a large quantity of them, and the other one is bovine brain.

I cannot really remember which is which, and which gave what results. That was about ten years ago or more.

DR. ROSS: Just a question. Is there any way of translating? You gave us the relative rates of incorporation from NAA compared with acetate as the gold standard. Can we translate that into rates in micromoles per gram or something which you might -DR. LEDEEN: Oh, it would be difficult to make that calculation because of the methodology. I can talk to you about that later if you like. It's difficult to do that.

DR. TRAPP: It appeared to me that the 57-kilodalton band is a little higher than that. Have you considered the possibility that incorporation of this enzyme into myelin in the membrane requires dimerization of the molecule? And are you potentially looking at a dimer, rather than a separate molecule?

DR. LEDEEN: That we're looking at a dimer? Well, then we should see some of the monomer, too, as well as the dimer if that's the case, shouldn't we? You might not expect it to. Well, maybe it could. Well, that is an interesting possibility, yes. So sometimes it's difficult to get a really accurate measure of molecular weights, but it's within the range anyway.

SESSION CO-CHAIR MATALON: I think this is a good question because this same question came up when we presented the data at another meeting; the Society for Inborn Metabolic Diseases. And the question came up, could this be a dimer? I have no answer for that, really. It could be.

DR. MADHAVARAO: In SDS-PAGE, unless they are covalently linked, you cannot expect to have a dimer, right?

PARTICIPANT: Or just to comment that it could be glycosylated, that the protein that went into myelination or in that component, could it be glycosylated?

DR. LEDEEN: In fact, we're looking at that. I should mention that my colleague who did a lot of this work is here, Dr. Wu. He is in the process of doing it. Unfortunately, we don't have a clear answer, but it would take too many carbohydrates to make that much difference in molecular weight.

But we know that some glycoproteins migrate aberrantly. They don't conform exactly to their true molecular weight after they acquire carbohydrate. So it's possible.

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