Unveiling a Hidden Biomarker of Inflammation and Tumor Progression: The 65 kDa Isoform of MMP-9 New Horizons for Therapy.

Cancer metastasis is a stage of the disease where therapy is mostly ineffective; hence, the need to find reliable markers of its onset. The metalloproteinase-9 (MMP-9, gelatinase B) in its 82 kDa active form, is a good candidate, but here we show that the correspondent little known 65 kDa active MMP-9 isoform, often misrepresented with the other gelatinase MMP-2, is a more suitable marker. Sera from patients with lung and breast cancer were analyzed by bidimensional zymography to detect the activity of MMP-9 and MMP-2. Enzyme identity was confirmed by comparison with MMP-9 standards and by western blotting. The 65 kDa isoform of MMP-9 is a suitable biomarker to monitor tumor progression from tissue neoplasms to metastatic stage, as its activity begins to appear when disease severity increases and becomes very high in metastasis. Moreover, the 65 kDa MMP-9, which derives from the 82 kDa MMP-9, no longer responds to natural MMP-9 inhibitors. As its activity cannot be controlled, its appearance may warn that the pathological process is becoming irreversible. Identification and inhibition of the enzymes converting the inhibitor-sensitive 82 kDa MMP-9 into the corresponding "wild" 65 kDa MMP-9 may allow to develop therapies capable of blocking metastases.

The human gut microbiota is neither an organ nor a commensal.

The recent explosive increase in the number of works on gut microbiota has been accompanied by the spread of rather vague or improper definitions, chosen more for common use than for experimental evidence. Among them are those defining the human gut microbiota as an organ of our body or as a commensal. But, is the human gut microbiota an organ or a commensal? Here, we address this issue to spearhead a reflection on the real roles of the human gut microbiota in our life. Actually, the misuse of the vocabulary used to describe the properties and functions of the gut microbiota may generate confusion and cause misunderstandings both in the scientific community and among the general public.

Undigested Food and Gut Microbiota May Cooperate in the Pathogenesis of Neuroinflammatory Diseases: A Matter of Barriers and a Proposal on the Origin of Organ Specificity.

As food is an active subject and may have anti-inflammatory or pro-inflammatory effects, dietary habits may modulate the low-grade neuroinflammation associated with chronic neurodegenerative diseases. Food is living matter different from us, but made of our own nature. Therefore, it is at the same time foreign to us (non-self), if not yet digested, and like us (self), after its complete digestion. To avoid the efflux of undigested food from the lumen, the intestinal barrier must remain intact. What and how much we eat shape the composition of gut microbiota. Gut dysbiosis, as a consequence of Western diets, leads to intestinal inflammation and a leaky intestinal barrier. The efflux of undigested food, microbes, endotoxins, as well as immune-competent cells and molecules, causes chronic systemic inflammation. Opening of the blood-brain barrier may trigger microglia and astrocytes and set up neuroinflammation. We suggest that what determines the organ specificity of the autoimmune-inflammatory process may depend on food antigens resembling proteins of the organ being attacked. This applies to the brain and neuroinflammatory diseases, as to other organs and other diseases, including cancer. Understanding the cooperation between microbiota and undigested food in inflammatory diseases may clarify organ specificity, allow the setting up of adequate experimental models of disease and develop targeted dietary interventions.

Differential Modulation of NF-κB in Neurons and Astrocytes Underlies Neuroprotection and Antigliosis Activity of Natural Antioxidant Molecules.

Neuroinflammation, a hallmark of chronic neurodegenerative disorders, is characterized by sustained glial activation and the generation of an inflammatory loop, through the release of cytokines and other neurotoxic mediators that cause oxidative stress and limit functional repair of brain parenchyma. Dietary antioxidants may protect against neurodegenerative diseases by counteracting chronic neuroinflammation and reducing oxidative stress. Here, we describe the effects of a number of natural antioxidants (polyphenols, carotenoids, and thiolic molecules) in rescuing astrocytic function and neuronal viability following glial activation by reducing astrocyte proliferation and restoring astrocytic and neuronal survival and basal levels of reactive oxygen species (ROS). All antioxidant molecules are also effective under conditions of oxidative stress and glutamate toxicity, two maladaptive components of neuroinflammatory processes. Moreover, it is remarkable that their antioxidant and anti-inflammatory activity occurs through differential modulation of NF-κB binding activity in neurons and astrocytes. In fact, we show that inflammatory stimuli promote a significant induction of NF-κB binding activity in astrocytes and its concomitant reduction in neurons. These changes are prevented in astrocytes and neurons pretreated with the antioxidant molecules, suggesting that NF-κB plays a key role in the modulation of survival and anti-inflammatory responses. Finally, we newly demonstrate that effective antigliosis and neuroprotective activity is achieved with a defined cocktail of four natural antioxidants at very low concentrations, suggesting a promising strategy to reduce inflammatory and oxidative damage in neurodegenerative diseases with limited side effects.

Molecular structure and function of myelin protein P0 in membrane stacking.

Compact myelin forms the basis of nerve insulation essential for higher vertebrates. Dozens of myelin membrane bilayers undergo tight stacking, and in the peripheral nervous system, this is partially enabled by myelin protein zero (P0). Consisting of an immunoglobulin (Ig)-like extracellular domain, a single transmembrane helix, and a cytoplasmic extension (P0ct), P0 harbours an important task in ensuring the integrity of compact myelin in the extracellular compartment, referred to as the intraperiod line. Several disease mutations resulting in peripheral neuropathies have been identified for P0, reflecting its physiological importance, but the arrangement of P0 within the myelin ultrastructure remains obscure. We performed a biophysical characterization of recombinant P0ct. P0ct contributes to the binding affinity between apposed cytoplasmic myelin membrane leaflets, which not only results in changes of the bilayer properties, but also potentially involves the arrangement of the Ig-like domains in a manner that stabilizes the intraperiod line. Transmission electron cryomicroscopy of native full-length P0 showed that P0 stacks lipid membranes by forming antiparallel dimers between the extracellular Ig-like domains. The zipper-like arrangement of the P0 extracellular domains between two membranes explains the double structure of the myelin intraperiod line. Our results contribute to the understanding of PNS myelin, the role of P0 therein, and the underlying molecular foundation of compact myelin stability in health and disease.

Diet, Gut Microbiota, and Vitamins D + A in Multiple Sclerosis.

Central to the understanding of the relationships between diet, gut microbiota, and vitamins D and A in multiple sclerosis is low-grade inflammation, which is involved in all chronic inflammatory diseases and is influenced by each of the above effectors. We show that food components have either proinflammatory or anti-inflammatory effects and influence both the human metabolism (the "metabolome") and the composition of gut microbiota. Hypercaloric, high-animal-fat Western diets favor anabolism and change gut microbiota composition towards dysbiosis. Subsequent intestinal inflammation leads to leakage of the gut barrier, disruption of the blood-brain barrier, and neuroinflammation. Conversely, a vegetarian diet, rich in fiber, is coherent with gut eubiosis and a healthy condition. Vitamin D levels, mainly insufficient in a persistent low-grade inflammatory status, can be restored to optimal values only by administration of high amounts of cholecalciferol. At its optimal values (>30 ng/ml), vitamin D requires vitamin A for the binding to the vitamin D receptor and exert its anti-inflammatory action. Both vitamins must be supplied to the subjects lacking vitamin D. We conclude that nutrients, including the nondigestible dietary fibers, have a leading role in tackling the low-grade inflammation associated with chronic inflammatory diseases. Their action is mediated by gut microbiota and any microbial change induced by diet modifies host-microbe interactions in a consequent way, to improve the disease or worsen it.

Anti-inflammatory nutritional intervention in patients with relapsing-remitting and primary-progressive multiple sclerosis: A pilot study.

The aim of this work was to assess the influence of nutritional intervention on inflammatory status and wellness in people with multiple sclerosis. To this end, in a seven-month pilot study we investigated the effects of a calorie-restricted, semi-vegetarian diet and administration of vitamin D and other dietary supplements (fish oil, lipoic acid, omega-3 polyunsaturated fatty acids, resveratrol and multivitamin complex) in 33 patients with relapsing-remitting multiple sclerosis and 10 patients with primary-progressive multiple sclerosis. At 0/3/6 months, patients had neurological examination, filled questionnaires and underwent anthropometric measurements and biochemical analyses. Serum fatty acids and vitamin D levels were measured as markers of dietary compliance and nutritional efficacy of treatment, whereas serum gelatinase levels were analyzed as markers of inflammatory status. All patients had insufficient levels of vitamin D at baseline, but their values did not ameliorate following a weekly administration of 5000 IU, and rather decreased over time. Conversely, omega-3 polyunsaturated fatty acids increased already after three months, even under dietary restriction only. Co-treatment with interferon-beta in relapsing-remitting multiple sclerosis was irrelevant to vitamin D levels. After six months nutritional treatment, no significant changes in neurological signs were observed in any group. However, serum levels of the activated isoforms of gelatinase matrix metalloproteinase-9 decreased by 59% in primary-progressive multiple sclerosis and by 51% in relapsing-remitting multiple sclerosis patients under nutritional intervention, including dietary supplements. This study indicates that a healthy nutritional intervention is well accepted by people with multiple sclerosis and may ameliorate their physical and inflammatory status.

Neuroprotection by Cocktails of Dietary Antioxidants under Conditions of Nerve Growth Factor Deprivation.

Dietary antioxidants may be useful in counteracting the chronic inflammatory status in neurodegenerative diseases by reducing oxidative stress due to accumulation of reactive oxygen species (ROS). In this study, we newly described the efficacy of a number of dietary antioxidants (polyphenols, carotenoids, thiolic compounds, and oligoelements) on viability of neuronal PC12 cells following Nerve Growth Factor (NGF) deprivation, a model of age-related decrease of neurotrophic support that triggers neuronal loss. Neuroprotection by antioxidants during NGF deprivation for 24 h was largely dependent on their concentrations: all dietary antioxidants were able to efficiently support cell viability by reducing ROS levels and restoring mitochondrial function, while preserving the neuronal morphology. Moreover, ROS reduction and neuroprotection during NGF withdrawal were also achieved with defined cocktails of 3-6 different antioxidants at concentrations 5-60 times lower than those used in single treatments, suggesting that their antioxidant activity was preserved also at very low concentrations. Overall, these data indicate the beneficial effects of antioxidants against oxidative stress induced by decreased NGF availability and suggest that defined cocktails of dietary factors at low concentrations might be a suitable strategy to reduce oxidative damage in neurodegenerative diseases, while limiting possible side effects.

Nutrition facts in multiple sclerosis.

The question whether dietary habits and lifestyle have influence on the course of multiple sclerosis (MS) is still a matter of debate, and at present, MS therapy is not associated with any information on diet and lifestyle. Here we show that dietary factors and lifestyle may exacerbate or ameliorate MS symptoms by modulating the inflammatory status of the disease both in relapsing-remitting MS and in primary-progressive MS. This is achieved by controlling both the metabolic and inflammatory pathways in the human cell and the composition of commensal gut microbiota. What increases inflammation are hypercaloric Western-style diets, characterized by high salt, animal fat, red meat, sugar-sweetened drinks, fried food, low fiber, and lack of physical exercise. The persistence of this type of diet upregulates the metabolism of human cells toward biosynthetic pathways including those of proinflammatory molecules and also leads to a dysbiotic gut microbiota, alteration of intestinal immunity, and low-grade systemic inflammation. Conversely, exercise and low-calorie diets based on the assumption of vegetables, fruit, legumes, fish, prebiotics, and probiotics act on nuclear receptors and enzymes that upregulate oxidative metabolism, downregulate the synthesis of proinflammatory molecules, and restore or maintain a healthy symbiotic gut microbiota. Now that we know the molecular mechanisms by which dietary factors and exercise affect the inflammatory status in MS, we can expect that a nutritional intervention with anti-inflammatory food and dietary supplements can alleviate possible side effects of immune-modulatory drugs and the symptoms of chronic fatigue syndrome and thus favor patient wellness.

Heterogeneity of serum gelatinases MMP-2 and MMP-9 isoforms and charge variants.

The matrix metalloproteinases (MMPs) gelatinase A (MMP-2) and gelatinase B (MMP-9) are mediators of brain injury in multiple sclerosis (MS) and valuable biomarkers of disease activity. We applied bidimensional zymography (2-DZ) as an extension of classic monodimensional zymography (1-DZ) to analyse the complete pattern of isoforms and post-translational modifications of both MMP-9 and MMP-2 present in the sera of MS patients. The enzymes were separated on the basis of their isoelectric points (pI) and apparent molecular weights (Mw) and identified both by comparison with standard enzyme preparations and by Western blot analysis. Two MMP-2 isoforms, and at least three different isoforms and two different states of organization of MMP-9 (the multimeric MMP-9 and the N-GAL-MMP-9 complex) were observed. In addition, 2-DZ revealed for the first time that all MMP-9 and MMP-2 isoforms actually exist in the form of charge variants: four or five variants in the NGAL complex, more charge variants in the case of MMP-9; and five to seven charge variants for MMP-2. Charge variants were also observed in recombinant enzymes and, after concentration, also in sera from healthy individuals. Sialylation (MMP-9) and phosphorylation (MMP-2) contributed to molecular heterogeneity. The detection of charge variants of MMP-9 and MMP-2 in MS serum samples illustrates the power of 2-DZ and demonstrates that in previous studies MMP mixtures, rather than single molecules, were analysed. These observations open perspectives for better diagnosis and prognosis of many diseases and need to be critically interpreted when applying other methods for MS and other diseases.

Peel LTP (Pru p 3)--the major allergen of peach--is methylated. A proteomic study.

Lipid transfer protein (LTP, Pru p 3) is the major allergen of peach (Prunus persica), and is in a greater abundance in the peel than in the pulp of the fruit. Peel LTP is more allergenic than pulp LTP, but it is not clear whether this is due to its specific allergenic properties or to its higher concentration. In this study, we have used a new one-step, rapid procedure for the purification of LTP from peel and pulp of four peach varieties [Gladys (white flesh), California (nectarine yellow flesh), Plusplus (yellow flesh), Red Fair (nectarine yellow flesh)] harvested in a field grown in Southern Italy. Purification was based on miniature reversed-phase chromatography, a procedure suitable for proteomic study. Proteomic analysis of purified LTPs revealed that the amino acid sequence of LTP was identical in all peach genotypes but, for the first time, peel LTP was found to be methylated.

What are the proteolytic enzymes of honey and what they do tell us? A fingerprint analysis by 2-D zymography of unifloral honeys.

Honey is a sweet and healthy food produced by honeybees (Apis mellifera L.) from flower nectars. Using bidimensional zymography, we have detected the, until now unrevealed, proteolytic activities present in row honey samples. The resulting zymograms were specific for each type of the four unifloral honey under study, and enzymes were identified as serine proteases by the use of specific inhibitors. Further, using bidimensional electrophoresis, we have shown that honey proteases are able to degrade the major Royal Jelly proteins and in particular MRPJ-1, the protein that promotes queen differentiation in honeybees. Our findings open new perspectives for the better understanding of honeybee development, social behaviour and role in honey production. The now discovered honey proteases may influence honey properties and quality, and bidimensional zymograms might be useful to distinguish between different honey types, establish their age and floral origin, and allow honey certification.

Digestive enzymes of the crustaceans Munida and their application in cheese manufacturing: a review.

Crustaceans Munida (fam. Galatheideae, ord. Decapodi) were fished in the Southern Adriatic Sea and their proteolytic activities were characterized and tested for potential application in cheese manufacturing. Enzymes extracted from whole crustaceans, mainly serine proteases, showed high caseinolytic and moderate clotting activities. Analysis by 2D zymography of the digestive enzymes extracted from Munida hepatopancreas, showed the presence of several isotrypsin- and isochymotrypsin-like enzymes in the range of 20-34 kDa and 4.1-5.8 pI. Moreover, specific enzymatic assays showed the presence of aminopeptidases and carboxypeptidases A and B. Overall, optimum activity was achieved at pH 7.5 and 40-45 °C. Caseinolytic activity, determined both spectrophotometrically and by SDS gel electrophoresis, indicated higher activity on ß-casein than on α-casein. Miniature cheddar-type cheeses and Pecorino-type cheeses were manufactured by adding starter, rennet and Munida extracts to milk. Reverse-phase HPLC and MALDI-ToF mass spectrometry showed a more complex pattern of proteolytic products in cheeses made using Munida instead of chymosin. Munida extracts were found to degrade the chymosin-derived ß-casein fragment f193-209, one of the peptides associated with bitterness in cheese. In conclusion, Munida digestive enzymes represent a promising tool for development of new cheese products and shorten cheese ripening when used either alone or in addition to calf rennet.

May diet and dietary supplements improve the wellness of multiple sclerosis patients? A molecular approach.

Multiple sclerosis is a complex and multifactorial neurological disease, and nutrition is one of the environmental factors possibly involved in its pathogenesis. At present, the role of nutrition is unclear, and MS therapy is not associated to a particular diet. MS clinical trials based on specific diets or dietary supplements are very few and in some cases controversial. To understand how diet can influence the course of MS and improve the wellness of MS patients, it is necessary to identify the dietary molecules, their targets and the molecular mechanisms involved in the control of the disease. The aim of this paper is to provide a molecular basis for the nutritional intervention in MS by evaluating at molecular level the effect of dietary molecules on the inflammatory and autoimmune processes involved in the disease.

2-D zymographic analysis of Broccoli (Brassica oleracea L. var. Italica) florets proteases: follow up of cysteine protease isotypes in the course of post-harvest senescence.

Zymographic analysis of Broccoli florets (Brassica oleracea L. var. Italica) revealed the presence of acidic metallo-proteases, serine proteases and cysteine proteases. Under conditions which were denaturing for the other proteases, the study was restricted to cysteine proteases. 2-D zymography, a technique that combines IEF and zymography was used to show the presence of 11 different cysteine protease spots with molecular mass of 44 and 47-48kDa and pIs ranging between 4.1 and 4.7. pI differences could be ascribed to different degrees of phosphorylation that partly disappeared in the presence of alkaline phosphatase. Post-harvest senescence of Broccoli florets was characterized by decrease in protein and chlorophyll contents and increase of protease activity. In particular, as determined by 2-D zymography, the presence of cysteine protease clearly increased during senescence, a finding that may represent a useful tool for the control of the aging process.

Structure-dependent inhibition of gelatinases by dietary antioxidants in rat astrocytes and sera of multiple sclerosis patients.

We investigated whether polyphenols modulate the expression and activity of the enzymes gelatinases A (MMP-2) and B (MMP-9), involved in the pathogenesis of multiple sclerosis (MS). LPS-activated primary rat astrocytes were treated with the flavonoids quercetin (QRC) and cathechins [green tea extract (GTE)] and the non-flavonoids resveratrol (RSV) and tyrosol/hydroxytyrosol (Oliplus). As assessed by zymography and RT-PCR, RSV and Oliplus, but not QRC and GTE, dose-dependently inhibited the LPS-induced levels and mRNA expression of MMP-2 and MMP-9. By contrast, in cell-free systems direct inhibition of gelatinase activity in MS sera was determined by QRC and GTE, but not by RSV. Oliplus was only partially effective. Our results indicate that the flavonoids and non-flavonoids tested exert their inhibitory effect on MMPs, displaying different mechanisms of action, possibly related to their structure. Therefore, their combined use may represent a powerful tool for the down-regulation of MMPs in the course of MS.

Analysis of green kiwi fruit (Actinidia deliciosa cv. Hayward) proteinases by two-dimensional zymography and direct identification of zymographic spots by mass spectrometry.

BACKGROUND: Proteinases present in kiwi fruits are potentially allergenic enzymes belonging to the papain family of cysteine proteinases. Actinidin is a prominent kiwi enzyme. The study of kiwi proteinases is important for the follow-up of fruit maturation, a deeper insight in the allergenic properties of individual proteins, and the application of kiwi proteinases for meat tenderisation and other industrial purposes. RESULTS: Kiwi crude extracts were analysed by two-dimensional zymography on gelatin-containing gels. The digestion by the reactivated proteolytic enzymes after electrophoresis resulted in insights into kiwi proteinases. A mixture of several enzyme isotypes with the same pI but different molecular mass was observed. Clear spots, corresponding to the proteolytic activities, were excised, digested with trypsin, and submitted to MALDI-ToF mass spectrometry for protein identification. The most representative enzyme was actinidin. CONCLUSIONS: The innovative achievements of the present study are the: (1) two-dimensional zymographic map of kiwi gelatinases without the need for extensive purification; and (2) direct identification of proteinase isotypes by means of direct MALDI-ToF MS analysis of the zymographic spots.

Developmental omega-3 supplementation improves motor skills in juvenile-adult rats.

Long-chain polyunsaturated fatty acids are critical for brain growth spurt during both foetal and postnatal period. They play important roles in the expression of genes regulating cell differentiation and neuronal growth, as well as in the development of synaptic processing of neural cell interaction. Foetus and placenta are dependent on maternal supply for their growth and development, and supplemented infants show significantly greater mental and psychomotor scores. In particular, it has been shown that if mothers take omega-3 supplements, their babies are smarter and better physically coordinated. On these grounds, the aim of the present study was to investigate, in the Sprague-Dawley rat, the effects of perinatal treatment with omega-3 on motor activity, motor coordination, motor learning and memory. From gestational day 8 throughout the lactation period, dams received either an emulsion of 0.05g/kg body weight omega-3 in fruit juice, or an emulsion of 1g/kg body weight omega-3 in fruit juice or just the fruit juice (control). Omega-3 formula was made of 27% docosahexaenoic acid and 53% eicosapentaenoic acid. On the day of birth (postnatal day 1), all pups were weighed, and then randomly culled to eight pups per litter. Pups were weaned at 21 days of age. One male pup per litter from each litter (control, n=6; omega-3 0.05g/kg, n=5; omega-3 1g/kg, n=6) was used. Both control and treated rats were tested for (i) locomotor activity using the open field paradigm, (ii) motor coordination and motor learning using the rotarod/accelerod task and (iii) memory using the passive avoidance paradigm. Rats were tested on postnatal day 21 and re-tested on postnatal day 90. As a result, docosahexaenoic acid and eicosapentaenoic acid supplementation significantly improved motor coordination. In particular, the latency to fall at the first speed was significantly increased in the treated rats as compared to the control animals. This benefit was observed with both doses at each tested age. The rat performance in accelerating rotation speed mode, which provides an indication of motor learning ability, was not modified by the omega-3 supply. Finally, the omega-3 treatment did not influence motor activity in the open field-tested rats, nor the memory ability in the passive avoidance task. In conclusion, perinatal omega-3 supplementation exerts a long lasting beneficial effect on the rotarod performance indicating improvement in balance and motor coordination and, possibly, in the functioning of pathways governing this task.

Cationic liposomes target sites of acute neuroinflammation in experimental autoimmune encephalomyelitis.

The binding selectivity of charged liposomes to the spinal cord of rats affected by experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, was investigated. Positively and negatively charged liposomes were injected into the tail vein of rats, and blood/brain barrier (BBB) targeting was determined by confocal microscopy as a function of the temporal evolution of the inflammatory response. Accumulation in spinal cord endoneural vessels was observed for cationic, but not for anionic, liposomes, and only in EAE but not in healthy rats. The overall binding efficacy paralleled the severity of the clinical score, but targeting was observed already before clinical manifestation of inflammation. Preferential binding of positively charged liposomes in the course of acute EAE can be ascribed to subtle changes of BBB morphology and charge distribution in a similar way as for the binding of cationic particles to proliferating vasculature in chronic inflammation and angiogenesis. Our findings suggest that vascular changes related to increased binding affinity for cationic particles are very early events within the inflammatory reaction in acute EAE. Investigation of cationic vascular targeting can help to shed further light on these occurrences, and, potentially, new diagnostic and therapeutic options may become available. In neuroinflammatory diseases, cationic colloidal carrier particles may enable intervention at affected BBB by an approach which is independent from permeability increase.

The different forms of PNS myelin P0 protein within and outside lipid rafts.

It is now well established that plasma membranes, such as the myelin sheath, are made of different microdomains with different lipid and protein composition. Lipid rafts are made mainly of sphingolipids and cholesterol, whereas the non-raft regions are made mainly of phosphoglycerides. Most myelin proteins may distribute themselves in raft and non-raft microdomains but the driving force that gives rise to their different distribution is not known yet. In this paper, we have studied the distribution of protein zero (P0), the most representative protein of PNS myelin, in the membrane microdomains. To this end, we have purified P0 from both non-raft (soluble P0, P0-S) and raft (P0-R) regions of PNS. Purified proteins were analyzed by two-dimensional gel electrophoresis and identified and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A detailed structural description of the two P0 forms is given in terms of amino acid sequence, post-translational modifications, and composition of associated lipids. Our findings suggest that structural differences between the two proteins, mainly related to the glycogroups, might be responsible for their different localization.