Commentary on two recent papers on GcMAF

 Commentary on two recent papers on GcMAF

Marco Ruggiero, MD, PhD

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Two important papers on GcMAF have been published in the past few days in peer-reviewed journals.

The paper by Borges and Rehder of the School of Molecular Sciences and of the Biodesign Institute at Arizona State University, Tempe, AZ, USA, clari es the chemical structure of the protein moiety of GcMAF, and represents a signifcant improvement in our understanding of the concepts and significance of GcMAF and nagalase. (Glycan structure of Gc Protein-Derived Macrophage Activating Factor as revealed by mass spec- trometry. Borges CR, Rehder DS. Arch Biochem Biophys. 2016 Aug 5. pii: S0003-9861(16)30278-8. doi: 10.1016/j. abb.2016.08.006).

It is interesting to notice that the paper by Borges and Rehder was published almost simultaneously with our latest paper on GcMAF (Medical Hypotheses 94 (2016) 126–131), and, although none of us was aware of each other’s imminent publication, the two papers are fully consistent with each other as they shed a new light on the interpretation of the results that have accumulated over the course of the past twenty years on GcMAF.

It is worth remembering that Borges and Rehder had published another important paper on the subject a few years ago, where they demonstrated that there is no lack of GcMAF precursor in cancer patients, as opposed to the prevailing theory of those days (Protein Sci. 2009 Oct;18(10):2036-42. doi: 10.1002/pro.214. Glycosylation status of vitamin D binding protein in cancer patients. Rehder DS, Nelson RW, Borges CR). Thus, according to the original theory rst proposed by Yamamoto, nagalase would “destroy” the precursor of Gc- MAF and, therefore, cancer patients would be unable to produce endogenous GcMAF: because of this, Gc- MAF had to be administered from the outside (Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF. Yamamoto N, Suyama H, Yamamoto N. Transl Oncol. 2008 Jul;1(2):65– 72).

Borges et al. demonstrated that this was not the case and cancer patients have plenty of their own GcMAF.

In the study of 2009 Borges et al. wrote: “The presence of relatively abundant quantities (5–10 mg/L or 100–200 nM) of what might nominally be considered GcMAF (i.e., DBP modi ed by a single terminal Gal- NAc residue) in cancer patient serum … present a paradox in which cancer patients with relatively high cir- culating concentrations of GcMAF can effectively be treated with trace quantities of (exogenous) GcMAF”.

This observation seems to indicate that there is no shortage of endogenous GcMAF production in cancer patients and, therefore, that GcMAF is not responsible for “protection” against the development of cancer.

Such a concept is further corroborated by the study of the cancer risk in individuals harboring the Gc2 alle- le only (Gc2 homozygotes) of the Gc protein. These individuals are unable to glycosylate the Gc protein on threonine 420 due to its substitution by lysine.

Thus, there is no N-acetylgalactosamine (GalNAc) in position 420. In other words, Gc2 homozygotes are unable to produce one single molecule of GcMAF but, despite this fact, the risk of cancer in these individu- als is decreased rather than increased as one would have expected given the absence of bona de GcMAF (Abbas S, Linseisen J, Slanger T, et al.: The Gc2 allele of the vitamin D binding protein is associated with a decrea- sed postmenopausal breast cancer risk, independent of the vitamin D status. Cancer Epidemiol Biomarkers Prev, 2008, 17: 1339-1343.). It should be noticed that this inconsistency refers to breast cancer that is one of the types of cancer where GcMAF had proven effective in vitro, and in vivo.

In the paper published a few days ago, Borges and Rehder further confirm these observations using carefully designed mass spectrometry methods, and highlight two important points that lend credit to our most recent hypothesis on the nature of GcMAF (Medical Hypotheses 94 (2016) 126–131).

  1. Nagalase does not affect the Gc protein, that is the precursor of GcMAF and, therefore, elevated nagalase levels do not correspond to reduced production of GcMAF. At variance with previous assumptions, Borges and Rehder demonstrate that nagalase is not endowed with endo-glycosi- dase activity and, therefore, cannot degrade the Gc protein. This carefully conducted observation explains the reason why cancer patients have elevated levels of endogenous GcMAF despite elevate serum nagalase activity.
  2. Beta-galactosidase, one of the enzymes used to (theoretically) convert the inactive Gc-protein into the active GcMAF by removing the galactose from the threonine 420 and exposing the GalNAc, does not work on the Gc-protein and does not remove the galactose; this means that the procedures to produce GcMAF by removal of galactose using beta-gaalcotsidase are, in actuality, ineffective. According to this observation, enzymatic treatment of purified Gc protein, or of serum, does not yield GcMAF but only a desialidated Gc protein where the GalNAc is still “covered” by galactose. According to the original theory proposed by Yamamoto, such a Gc protein with galactose still attached to GalNAc should be devoid of macrophage stimulating activity.

The results published a few days ago by Borges and Rehder are fully consistent with those published in 2010 by a Danish group using mass spectrometry, demonstrating that the three sugars on the Gc protein are arranged in a linear fashion (threonine 420-GalNAc-galactose-sialic acid) and, therefore, beta-galac- tosidase cannot work since it is not endowed with endo-glycosidase activity (Biochim Biophys Acta. 2010 Apr;1804(4):909-17. doi: 10.1016/j.bbapap.2009.12.022. Epub 2010 Jan 13. The glycosylation and characteriz- ation of the candidate Gc macrophage activating factor. Ravnsborg T1, Olsen DT, Thysen AH, Christiansen M, Houen G, Højrup P).

The lack of effect of the beta-galactosidase also forces to reconsider the interpretation of the results reported by the researchers from the University of Tokushima and from the Saisei Mirai Clinic of Japan, who demonstrated anti-cancer effects obtained by treating the Gc protein only with beta-galactosidase (Anti- tumor effect of degalactosylated gc-globulin on orthotopic grafted lung cancer in mice. Hirota K, Nakagawa Y, Takeuchi R, Uto Y, Hori H, Onizuka S, Terada H. Anticancer Res. 2013 Jul;33(7):2911-5. β-Galactosidase treatment is a common rst-stage modi cation of the three major subtypes of Gc protein to GcMAF. Uto Y, Yamamoto S, Mukai H, Ishiyama N, Takeuchi R, Nakagawa Y, Hirota K, Terada H, Onizuka S, Hori H. Anticancer Res. 2012 Jun;32(6):2359-64).

Quite obviously, treatment with beta-galactosidase alone did not produce any bona de GcMAF and, therefore, the observed anti-cancer effects have to be ascribed to some other mechanism or some other molecules.

The two most recent papers published on GcMAF may thus help reconcile several contradicting observations that have accumulated over the years. Such inconsistencies and contradictions are:

  1. The procedure to produce GcMAF using enzymatic treatment of purified Gc protein or serum using beta-galactosidase does not produce one single molecule of bona de GcMAF.
  2. Cancer patients who were successfully treated with what was thought to be GcMAF, but in actuality was not, had plenty of their own GcMAF, a quantity that was far higher than the trace amounts of the desialidated Gc-protein that was administered thinking it was GcMAF. Although the levels of GcMAF in autistic patients has never been determined, the inability of nagalase to reduce the production of GcMAF leads to conclude that also autistic children with elevated nagalase had normal level of endogenous GcMAF. Nevertherless they were successfully treated with desialidated Gc-protein that was administered thinking it was GcMAF.
  3. These inconsistencies notwithstanding, it is a fact that “GcMAF” (between quotation marks since now we know that it was not bona de GcMAF) has proven effective in vitro and in vivo, and the laboratory and clinical data obtained with “GcMAF” have been repeatedly and independently confirmed.

These contradictions and inconsistencies may all be solved if we consider the hypothesis put forward our most recent paper in Medical Hypotheses; a hypothesis that reconciles the apparently contradicting observations quoted above. In other words, we do not dispute the validity of any of results of the papers thus far published on GcMAF; in our opinion, it is their interpretation that needs to be revisited at the light of the most recent publications.

Our concept, that has been defined by Borges as “interesting and plausible” at the light of their own most recent results, is that the Gc protein, whether with one, two or three sugars, or with any sugar moiety as in the case of the Gc2 homozygotes, plays a little role, if any, in the activation of macrophages or in any of the other biological and clinical effects that we and others have described for what we thought was “GcMAF”. According to our hypothesis, it is a glycosaminoglycan that binds to the Gc protein, chondroitin sulfate, in conjunction with vitamin D and oleic acid bound to the Gc protein, that is responsible for the effects attributed to the “GcMAF”. The Gc protein, deglycosylated or not, at most is a carrier for chondroitin sulfate, vitamin D and oleic acid.

Our new interpretation also explains the well acknowledged fact that treatment with “GcMAF” leads to reduction of nagalase levels in patients with different diseases, and leads to reconsider the role of nagalase as a marker.

Thus, nagalase cannot be considered a marker of immunodeficiency since its elevated levels do not correspond to decreased levels of endogenous GcMAF and, more importantly, elevated levels of nagalase have never been associated with any deficiency of the immune system. For example, autistic subjects have levels of nagalase higher than those of many cancer patients and nevertheless they are not immune deficient in the least.

However, nagalase seems to be a very reliable marker of chronic inflammation regardless of the causes that are responsible for in ammation (J Proteome Res. 2015 Aug 7;14(8):3123-35). This would explain the reason why nagalase is elevated in diseases that have nothing in common but chronic inflammation such as cancer and autism.

In the case of cancer, it is of particular importance to notice that in ammation in itself plays a major role in the progression of the disease and it is, in and by itself, a predictor of negative prognosis as we have de- monstrated in 2012 (Fabris et al. American Journal of Immunology, 2012, 8 (3), 65-70).

If we consider that the biological and clinical effects that had been attributed to GcMAF are indeed to be ascribed to a multimolecular complex made of chondroitin sulfate, vitamin D and oleic acid, with the Gc protein playing a minor role if any, then it makes full sense these three molecules decrease nagalase, since they are very well known to have anti-inflammatory properties. It is also worth mentioning that chondroitin sulfate, vitamin D and oleic acid also show immune-stimulatory, anticancer and neuroprotective pro- perties that are superimposable to the biological and clinical properties thus far attributed to GcMAF.

Therefore, nagalase could be interpreted as a marker of chronic inflammation and evaluation of serum nagalase levels could be useful in monitoring the effectiveness of immunotherapies regardless of the under- lying disease. In this respect, nagalase measurement would have an advantage over specific markers such as tumor markers, since its variations would be useful in a much broader range of conditions.

In conclusion, the almost simultaneous publication of the papers by Borges and Rehder and by our research group opens a completely new perspective in the field of immunotherapy and GcMAF research, and helps solving the inconsistencies and contradictions that have characterized this field in the recent past.

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