Enzyme Replacement Therapy On Gaucher Disease Biology Essay

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Gaucher Disease is the most common lysosomal storage disorders and is caused by the mutation of the gene encoding the enzyme glucocerebrosidase (GCase) leading to the accumulation of its substrate glucocerebroside mainly in macrophages. GCase is an enzyme with 497 amino acids that catalyse the hydrolysis of glucocerebroside into β-glucose and ceramide by cleaving the β-glycosidic bond. Clinical manifestations of GD include hepatosplenomegaly, musculoskeletal disorders and central nervous system (CNS) dysfunctions. It is an orphan disease that is inherited in an autosomal recessive manner. Over 200 mutations are identified in GD patients but the genotype-phenotype correlations are not well established. Enzyme Replacement Therapy (ERT) with imiglucerase, the DNA recombinant human GCase, is the standard of treatment in GD since its approval in 1994 due to its excellent efficacy and safety profile but more sources of GCase including human gene activation GCase (velaglucerase alfa) and plant-derived recombinant GCase (prGCD) are now available with the hope of increase the cost effectiveness of ERT. Finally, this review will discuss some active ongoing investigations on issues related to the disease and its treatments.


Gaucher Disease (GD) belongs to a group of metabolic disorders known as Lysosomal Storage Disorder (LSD) and is the most common disease in this group in which there is a deficiency of certain lysosomal enzymes or disturbance of lysosomal protein function. [1, 2, 3, 4] In Gaucher disease, the insufficient enzyme activity of lysosomal hydrolase glucocerebrosidase (Acid-β -Glucosidase, GCase), leads to the accumulation of its substrate glucocerebroside. [1, 2, 5] Glucocerebroside is catabolised product of glycosphingolipds. Apart from maintaining eukaryotic cell membrane integrity, glycosphingolipids also play an important role in intracellular signalling pathways and cell recognition.[2] Accumulation of glucocerebroside occurs predominately in macrophages, which are most prominent in liver, spleen, bone marrow and lymph lodes. [1, 2, 6] Gaucher cells refer to macrophages that accumulated with glucocerebroside. [1,6]

GD patients often share some common clinical manifestations including hepatosplenomegaly, musculoskeletal disorders and central nervous system (CNS) dysfunction (in type 2 and 3 patients). There is always no single theory to explain the link between accumulation of glucocerebroside and the pathologies. Glucocerebroside accumulation in the liver and spleen causes enlargements of these organs and is associated with early satiety and abdominal discomfort. [1,2,3,4] Splenomegaly is often associated with hypersplensim which will lead to anaemia and thrombocytopenia , therefore fatigue and bleeding are common presenting symptoms in GD patients.[1,3,4,6] Moreover, activated macrophages and more severe liver involvement may cause clotting factor abnormalities. [3] The musculoskeletal manifestations are caused by infiltration of Gaucher cells into the bone marrow. [1, 4, 5] It is believed that marrow expansion with subsequent vascular occlusion and compression and increased intraosseous pressure are caused by the infiltration of Gaucher cells, but the exact mechanism remains unclear. [4, 6] Moreover, it is suggested that release of cytokines will contribute to the high rates of bone turnover and failure of remodelling. [4] Common skeletal features include osteonecrosis, osteopenia, Erlenmyer flask deformity and bone fractures, all result in pain and disability to the patients. [1,4,5,6] Liver function is usually well preserved and pulmonary hypertension or interstitial lung disease caused by infiltration of Gaucher cells into lungs are less common.[1,4, 5,6] Clinical manifestations might also be contributed by the immune response but the relationship between glucocerebroside accumulation and production of inflammatory mediators is not well understood. [2, 5, 6] The pathology of neuropathic form of the disease is comparatively less well known. It is believed that neurones that accumulate with glucocerebroside are more sensitive to neurotoxins such as glutamate as a result of increased release of calcium from the endoplasmic reticulum (ER). [1, 2]

Gaucher disease is divided into three subtypes according to the rapidity of onset and the involvement of the CNS. Type 1, also known as the non-neuronopathic disease is the most common form of GD and in addition, the most common form of lysosomal storage disorder. Type 1 GD has a particular preference among the Ashkenazi Jewish origin where incidence is 1 in 850 live births and a carrier frequency of 1 in 10 to 1 in 17, while in the general population the incidence rate is an estimate of up to 1 in 60,000 live births. [1, 5, 6]

Type 2 GD or acute neuronopathic GD, is rapidly progressive and life-threatening. It is formerly known as infantile Gaucher disease in which babies appear normal at birth but symptoms develop by 6 months of age. It is characterised by bulbar involvement leading to difficulties in swallowing and aspiration of food and secretions into lungs. The vast majority of these children die by the age of 2 due to recurrent pneumonia or infection. In addition, hepatosplenomegaly and lung involvement are often observed. [7]

Type 3 GD or chronic neuropathic GD is subdivided into 3 variants. Type 3b has an earlier onset hepatosplenomegaly but slowly progressive neurologic involvement. [7] CNS involvement can include abnormal eye movement and slowing of development. On the other hand, Type 3a has an onset at late teens or early twenties with more rapidly progressive CNS deterioration but comparatively less involvement in visceral organs. [7] Type 3c is a newly identified variant in which progressive and fatal calcifications of the heart valves and aorta occur. [7]

The Enzyme, Glucocerebrosidase

Glucocerebrosidase (Acid -β-glucosidase, GCase) is a 497 amino acid long membrane glycoprotein found in lysosomes and belongs to the retaining glycoside hydrolase family 30. [8, 9, 10, 11]It cleaves the β-glucosidic bond of its naturally occurring substrate, glucocerebroside (also known as glucosylceramide) to give out glucose and ceramide as products. [8,10,11] Optimal hydrolytic activity is achieved with negatively charged lipids (phospholipids), hydrophobic agents, acidic pH and an enzyme activator, saposin C which interact with the phospholipids vesicles through reversible membrane binding. [8]The exact mechanisms of activation by the negatively charged phospholipids and/or saposin C are not fully known but it is suggested that the enzyme and saposin C molecule form an aggregate that would interact with the vesicular surface containing the substrate, hence facilitating the interaction of the enzyme and its substrate.[9] The catalytic cycle proceed via a two-step, double displacement mechanism that includes glucosylation of the active site by the substrate followed by deglucosylation of the enzyme with the release of β-glucose. [8, 10] During glucosylation, E340 acts as the nucleophile and attacks the O-glycosidic anomeric linkage at C1 of the substrate. [8, 10] This is followed by E235 acting as the presumptive acid/base and protonate the ceramide-glucosidic bond, with the formation of a partially stabilized oxycarbonium intermediate. The glucose moiety then becomes covalently attached to the E340 residue of the enzyme while the ceramide is released. [8] Another mechanism of glucosylation suggests a steric hindrance of E235 on the E340 nucleophilie attack and that a proton donation from E235 to the anomeric carbon of glucose produces a carbenion which is then attacked by the E340 nucleophile for glucosylation. [8] In the deglucosylation step, the enzyme-glucosyl complex is attacked by water and the reaction is base-assisted from the acid/base. β-glucose is released while regenerating the acid of the acid/base and the nucleophile. [8, 10]

Fig1: GCase catalyse the hydrolytic reaction by attacking the β-glycosidc bond of glucocerebroside and giving out β-glucose and ceramide. R, R1 = (CH2)13-21CH3. Figure modified from ref. [11].





Fig 2. Double displacement mechanism of GCase hydrolysis of its substrate. E340 act as the nucleophile while E235 act as putative acid/base. Acid of the E235 residue attack the anomeric carbon of the substrate and a partially stabilized oxycarbonium structure is formed. Ceramide (ROH) is released and a covalent glucosyl-enzyme structure is formed with the nucleophile E340. Water that is base-assisted from the acid/base then attacks the complex, giving out β-glucose and regenerates the E235 and E340 residues. Figure taken from ref. [8].

3D structure of GCase was resolved by X-ray diffraction crystal analysis and revealing that the enzyme comprises three folding domains. Domain I consists of a three-stranded antiparallel β-sheet that is flanked by a loop and a perpendicular amino-terminal strand. [9, 11] Domain II is an immunoglobulin (Ig) like fold formed by two closely associated β-sheets. [9, 11] Domain III is a (β/α)8 TIM barrel which contains the catalytic site.[9,11] Domain II and III seem to be connected by a flexible hinge while domain I and III are tightly interact with each other. [11]

Fig 3.The refined X-ray structure of GCase. Domain I, II and III are shown in magenta, green and blue respectively. The six most common GCase mutations are shown as ball in which red balls represent mutations causing severe disease (type 2 or 3 GD) while yellow balls represent mild disease(type I GD). Figure taken from ref. [11] xray.jpg

Fig.4 Distribution of single amino-acid substitution that lead to Gaucher disease in three-dimensional structure of GCase. Mutations scattered throughout the enzyme. Red balls represent mutations causing severe disease while yellow balls represent mild disease. Blue balls represent mutations that lack of clinical data to show the severity of the disease. Figure taken from ref.[11].

Glucocerebrosidase Gene Mutation

The gene encoding GCase is located in chromosome 1q21. [9, 12] Over 200 mutations have been identified in the glucocerebrosidase gene of GD patients, and the mutations are scattered throughout the enzyme. [10] The genotype-phenotype correlations of the disease have not been well established yet. Most of the mutations identified in GD are heteroallelic. [10,12] Since it is the combination of both mutated alleles that is important in defining the phenotype, this hetreoallelic characteristic make it more difficult to refine genotype-phenotype correlations. In addition, mutated GCase are degraded in the ER before reaching the lysosome via cellular quality control mechanism. [13, 14] This endoplasmic reticulum- associated degradation (ERAD) may alter the amount of intralysosomal enzyme and result in phenotypic variability.[13] There was experiment data supporting the idea that the severity of the disease is directed correlated to the extent to which GCase is retained in the ER and degraded via ERAD. [14] Moreover, macrophage is not the only cell type that accumulation of glucocerebroside occurs. Stored intralysosomal glucocerebroside may be transferred to other cell types which lead to dysfunction of multiple cell types and hence patients would present with different phenotypes. [13] Effect of enhancers and other genes located close to GCase gene could possibly affect phenotypic variability. One example is the Metaxin gene which is transcribed in reverse direction of the glucocerebrosidase pseudogene. [13] Mutation of metaxin has shown to affect embryonic development in animal model but the linking of this mutation to GD phenotype is not known. [13]

N370S is one of the few mutations that with identified genotype-phenotype correlation.N370S mutation is the most common mutant genotype in type 1 Gaucher disease, accounting for ~70% of disease alleles in Ashkenazi Jewish patient and ~44% of non-Jewish patients. [11, 12, 13, 14] The presence of at least one N370S alleles appears to be protective against early onset of Gaucher-related CNS dysfunction. [8, 13] N370 is located at the α-helix 7 of the TIM barrel which is at the interface between the TIM barrel and the Ig like domain. [11] This residue is not in close approximation to the active site so initial studies suggested it has little effect in active site function. [8] Further studies revealed that although N370 has no obvious direct interaction at the active site due to its distance from the active site, N370S mutations can cause local conformational effects which prescribe specific alignments in the active site and substantially alter the catalytic cycle.[8] The fact that N370S mutation is a residue specific but not positional effect is shown by substituting Asparagines with other amino acids (other than Serine) and result in a decrease in catalytic power. [8, 10] Furthermore, disturbance of the glucosylation and deglucosylation steps in the catalytic cycle is only seen with mutating N370 residue to Serine but not other amino acids. [8]

Another mutation that with identified genotype-phenotype correlation is the L444P, which predispose to severe neuropathic form of GD. [11, 12, 14] The L444 residue is located in the hydrophobic core of the Ig-like domain and mutation of L444 to proline would disrupt the hydrophobic core and lead to a local conformation change and hence an unstable enzyme.[11] Moreover, mutation of D409H was found to be in association with type 3c of GD in which calcification of cardiac valves are presented. [9, 12] Frankly speaking, mutations that result in neuropathic form of GD are usually more dramatic in their effects on enzyme folding and stability of the enzyme. [11, 14]

Enzyme Replacement Therapy

Enzyme replacement therapy (ERT) refers to replacing the mutated enzymes with exogenous enzymes that are functioning. Before the introduction of ERT, there were no specific treatments for GD and managements were solely of treating the complications and symptoms as well as supportive care. ERT has served as a gold standard treatment for GD since its approval and has become a model for treating other lysosomal storage disorders. [1] The first generation of ERT in GD involved the remodelling of GCase purified from human placenta cells, alglucerase (Ceredase). [15, 16] Imiglucerase (Cerezyme), approved in 1994, is produced by DNA recombinant technology from Chinese hamster ovary cells. [1, 16] The Gene-activated human GCase, Velaglucerase alfa has newly been approved by FDA (U.S. Food and Drug Administration) in 2010 [17] while the plant cell derived recombinant GCase (prGCD) is now on phase III clinical trial. [18]

Imiglucerase (Cerezyme)

Since the accumulation of glucocerebroside occurs mainly in macrophages, the aim of ERT is to target and deliver exogenous GCase into macrophages. The discovery of mannose receptors on macrophages led to the suggestion of exogenous GCase with mannose terminals could help to improve targeting and internalization by the macrophages [19]. Four of the five N-glycosylation sites (N19, N59, N146 and N270) are involved in co-translational glycosylation in which this glycosylation is essential for the production of an active GCase [10, 15]. Native GCase contains oligomannose on one of the four glycosylation sites but this is not sufficient enough for targeting mannose receptors on macrophages [19]. Therefore, remodelling of the enzyme is required to replace the oligosaccharides in native GCase with oligomannose [16, 19, 20]. Alglucerase and imiglucerase have three and four of the glycoslation sites remodelled to mannose core respectively and both have great improvement in targeting macrophages [19]. As alglucerase was isolated from human placenta, in theory it could be contaminated by pathogenic microorganisms. [16] For this reason, alglucerase was subsided after the introduction of imiglucerase [1, 16]. In the ER of mammalian cells, glycans are added to the N-linked glycosylation sites as high mannose structures and subsequently the complexes are trimmed back to Man5GlcNAc2 (Man, Mannose; GlcNAc, N-acetylglucosamine) by glycosidase in the ER and Glogi [19]. The complex oligosaccharide structures are then formed after the addition of GlcNAc, galactose and sialic acid [19]. Remodelled GCase with terminal mannose residues could be formed by using glycosidase inhibitors to block the addition of the first GlcNAc to the Mannose-3-core [19]. Accordingly, the predominant glycoform in imiglucerase is Man3GlcNAc2 with core fucoslyation [20, 21]. Apart from mannose, other glycoforms such as fucose, galactose and mannose-6-phosphate (M6P) are also formed in trace amount [19, 21]. Imiglucerase contains a single point mutation, R495H, when compare with the native GCase which is caused by a cloning artefact [1, 15, 19, 21]. This single amino acid substitution has shown no effect on the structural or biochemical properties of the enzyme [10, 19].

Fig.5 Major glycan structure of GCase expressed in Chinese hamster ovary cells before and after glycan remodelling. Figure taken from ref. [20].

Imiglucerase: Dosage Regimen

Imiglucerase is administrated as intravenous infusion over 1-2 hours. The dosage regimen should be individualised for every patient since patients respond to a variety of doses but a dosage of 60U/kg every two weeks could be used as a reference. [1, 16, 22, 23] A randomized trial had shown comparable results in the efficacy and safety of imiglucerase infusion every four weeks versus every two weeks at the same total monthly dose. [24] It brings hope to broaden dosage regimen options and improve patients' compliance by reducing the infusion frequency. Nonetheless, this study was only conducted on adult patients with stable type 1 GD. [24] On the other hand, there is an argument that low-dose; high-frequency infusion could lead to more efficient uptake of the GCase by macrophages and reports had shown satisfactory response when similar total monthly doses are administrated but this regimen is not widely used as treatment failure and non-compliance issues had been reported. [1] Unfortunately, it is difficult and complicated to compare different dosage regimen due to a range of variables among patients [1]. Despite the fact that some studies showed comparable improvements in haematological and visceral factors with the use of either high or low dosage regimen, it is well documented that skeletal disorders require higher dose for satisfactory improvements.[1, 6, 23]

Imiglucerase: Clinical Efficacy

Therapeutic goals of ERT include reduction of spleen size to less than eight times of normal; liver volume reduced to 1-1.5 times of normal; platelets counts increased to above 120 x 109/L or doubled if initial values were below 60x109/L; haemoglobin levels increased to ≥11g/dl and ≥12g/dl for women and men respectively; reduction of bone pain; and reduction in bone crises.[1,6,7] Improvements in haematological parameters (liver and spleen size, haemoglobin and platelet levels) usually become apparent after 6-12 months of initiation of treatment with a fast initial effect, followed by a slower improvement after the first 6 months. [1, 6, 23] Skeletal improvements occur much slower. [ 1,6,16,23] Some studies argued that whether ERT has direct impacts on the bone symptoms [7] although other studies have already provided evidence of normalization of bone marrow fat content, increased bone density and reduction of bone pain and bone crises.[1,6,16] A recent study using the data from the International Collaborative Gaucher Group (ICGG) in 195 GD patients receiving imiglucerase showed that the percentage of patients meeting specific therapeutic goals increased for all parameters at four years. [25] Moreover, proportion of patients that met all the six goals increased from 2.1% before treatment to 41.5% at four years.[6,25] This study also concluded that on average, patients receiving a higher dose would achieve more therapeutic goals. [25]

Fig. 6. Cumulative achievement of therapeutic goals at time of initiation and 4 years after initiation of imiglucerase. Data obtained from a study of 195 type 1 GD patients who enrolled in the ICGG Gaucher Registry. Figure taken from ref. [25].

A wide range of therapeutics goals are also being studied, including quality of life, pulmonary involvement and catching up of growth in children but they are harder to quantify by statistical means [6, 23] The clinical efficacy of using ERT in neuropathic form of the disease is in doubt. [1, 6, 23] Patients with type 3 GD show improvements in haematological and visceral aspects but the neurological progression do not seem to be halt or slowed down because the enzyme is too large to cross the blood brain barrier. [1,16,23] It is the same situation in type 2 of the disease and there is now a clear agreement that ERT is not indicated for type 2 GD. [16] A high dose of 120-240U/kg every 2 weeks has been introduced in treating type 3 GD but there was no solid evidence to support its efficacy of being superior to the standard dose. [1, 23]

Imiglucerase: Pharmacokinetics

Little is known about the tissue and cellular distribution and pharmacokinetics of infused ERT due to the lack of animal model that is analogous to human gaucher cells. [1, 6, 23] Serum half life of imiglucerase is 3.6 -10.4 minutes. [22, 23] Steady state enzymatic activity is reached within 30 minutes of infusion. [22, 23] Iodine-123-labelled recombinant enzyme was used to study the uptake and tissue distribution of the enzyme in 8 patients with type 1 GD [1,6,23] Uptake into liver and spleen reach peak concentration in 15-30 minutes.[6,23] 30% of the injected dose is taken up by the liver, 15% by the spleen and a significant amount is diffused into the bone marrow. [1,6,23] Clearance by the liver and spleen is biphasic, with a half life of 1-2 hours in the rapid phase and followed by a slower phase with half life of 34-42 hours.[1,6,23] The bone marrow mean half life was 14 hours. [1, 23] A lower dose, higher frequency dosage regimen was suggested as a result of the observation that the macrophages mannose-receptor mediated uptake is a saturable process. [1] Moreover, it is suggested that GCase binds to the 'classic' monocyte/macrophages mannose receptor as well as a distinct lower affinity mannose-dependent receptor that is widely present on the surface of a range of cell types including hepatocytes and endothelia cells. [1, 6] Some scientists, therefore, suggested that a lower concentration of GCase would favour binding to the classic receptor and so lead to a higher yield to the Gaucher cells. [1, 6] Nonetheless, as mentioned above, this low dose, high frequency regimen is not widely accepted.

Imiglucerase: Safety

Imiglucerase has an excellent safety profile and is well tolerated. An approximate of 13.8% of patients experienced adverse events after administration of imiglucerase [1,22,23] The most frequently reported adverse event is infusion- related reactions, such as chills, discomforts, rash, pruiritus, swelling at the injection site, each occurs in <1.5% of total treated patient [22, 23] Hypersensitivity reactions have been reported in about 6.6% of patients, symptoms include pruritus, flushing, urticaria, chest discomfort, dyspnea, coughing and hypotension.[1, 22] Anaphylaxis and other IgE related-reactions have been found in less than 1% of patients. [1, 22] In general, pre-treatment with corticosteroids and/or antihistamines and slower the infusion rate would allow patients to continue imiglucerase treatment. [1, 22, 23] Around 15% of patients developed IgG antibodies to imiglucerase during first year of treatment, however, only 46% of these patients experienced adverse events. [1, 22] Moreover, 90% of patients whom developed IgG antibodies become tolerate over time. [1, 23] In a nutshell, development of IgG antibodies seldom interferes with the use of imiglucerase. [1, 22, 23]

A double-blind, randomized trial was conducted and found that there were no significant difference in biological activity, pharmacological properties and safety profiles between Alglucerase and Imiglucerase. [1, 16, 19]

Gene Activated Human Glucocerebrosidase: Velaglucerase alfa

The gene activated human GCase, velaglucerase alfa, consist of an amino acid sequence that is identical to native human GCase since it is produced in the human cell line. [21, 23, 26] In gene activation, a promoter that activates the endogenous GCase gene is targeted by recombinant technology. [21] The four of the five N-glycoslyation sites are remodelled to high mannose- type glycans with six to nine mannose units using kifunensine, a mannosidase I inhibitor during cell culture. [19, 21, 26] Mannosidase I is responsible for the reduction of Man9GlcNAc2 to Man5GlcNAc2 during natural processing of GCase in the ER and/or Glogi, hence inhibiting its action would lead to GCase with primarily Man9GlcNAc2 oligomannose residues [19]. There is an argument that the higher mannose type glycan (nine mannose units) in velaglucerase alfa could promote internalization of the enzyme by macrophages (cf. three mannose units in imiglucerase).One study compared the internalization of imiglucerase and velaglucerase alfa by macrophages and demonstrated that internalization of velaglucerase alfa is 2.5fold more efficient than that of imiglucerase [21] On the other hand, another study comparing recombinant GCase of different chain lengths of oligomannose (Man 2 to Man 9) residues showed no significant difference in uptake by macrophages although difference in affinity to mannose receptor was observed. [19] Nonetheless, this study also demonstrated GCase with longer oligomannose chain length has higher affinity to serum mannose-binding lectin (MBL) [19]. This raise concern of using GCase with larger oligomannose structure since MBL is a collectin naturally found in the serum and binding of MBL can activate the innate immune system leading to activation of complement system, opsonisation and phagocytosis [19]. In spite of the fact that mannose receptor and MBL both bind to mannose residue and have similar carbohydrate recognition specificity, their difference in binding affinity, thus selectivity could be explained by the distinction of their overall conformation [19]. A study on the pharmacokinetics profile of Velaglucerase alfa showed that it has a comparable serum half life and clearance with imiglucerase and its safety profile is also consistent with that of imiglucerase. [26] The use of GCase with higher-mannose type glycans for ERT to improve clinical efficacy by improving its uptake by macrophages is yet to be confirmed, however, the approval of Velaglucerase alfa does provide an alternative treatment option and overcome the shortage of imiglucerase supply.

Recombinant Plant-Derived Glucocerebrosidase (prGCD)

The plant-derived recombinant human GCase (prGCD) is an alternative method to produce exogenous GCase. Currently, a GCase expressed in carrot cells is undergoing Phase III clinical trials. As discussed above, terminal mannose residues are important in targeting and internalization by macrophages and both imiglucrease and velaglucerase alfa require in vitro remodelling to produce terminal oligomannose residues. Terminal mannose residue is naturally occurring in GCase expressed in carrot cells, thus in vitro post-production remodelling is not required. [20, 27] In vivo generation of the terminal mannose seems to be caused by a special vacuolar enzyme, hence GCase expressed in carrot cells require targeting to this storage vacuole. [20] In order to achieve efficient expression on carrot cells and sufficient activity of the product, several modifications in the native human GCase gene has been made. For example, replace the signal peptide within the GCase gene with a basic endochitinase gene to improve translocation into the ER; and fuse the vacuolar signal sequence with the C-terminal of the GCase gene to facilitate storage vacuole targeting. [20]


Ω enhancer

Signal Peptide

Human GCase gene

Vacuolar signal


Fig.7. GCase gene used in the plant expression system. Figure modified from ref. [20].

X-ray crystal analysis revealed that prGCD has a structure that is highly similar to that of imiglucrease and the enzymatic activity of prGCD is also comparable [20,27]. Phase I clinical trials on healthy volunteers have confirmed there were no significant innate or humoral immune reactions after administration of prGCD while pre-clinical toxicological studies on primates also supported these results.[27] Meanwhile, there were conflicting reports demonstrating that 50% and 25% of non-allergic blood donors have developed specific antibodies for core xylose and core α-(1,3)-fucose respectively,[20] while these residues are present on prGCD but not imiglucerase nor velaglucerase alfa. [21] Whether the development of these antibodies would limit the use of plant-derived biopharmaceutical glycoproteins remains to be determined but it is obvious that the prGCD has several advantages over imiglucerase. [20, 27] prGCD production does not require the costly in vitro remodelling as prGCD naturally possess terminal mannose residues [20, 27]. Furthermore, production of recombinant glycoproteins in mammalian cells are generally more expensive and potentially less safe as mammalian derived components can be present in the manufacturing process [20, 27]. Another advantage of plant-derived recombinant glycoproteins is that they allow high batch-to-batch reproducibility and precise control over the growth process of the plant cells. [20, 27]

Ongoing Investigations


In order to evaluate and monitor the disease progression and patients' responses to therapeutic interventions, biomarkers are essential. Biomarkers are generally chemical entities and ideally they should provide indirect assessment of the disease activity but should directly correlate with disease severity [16, 28]. Moreover, the range or concentration of a biomarker should have no overlap between untreated patients and healthy individuals [29]. In the case of LSD, there are two potential classes of biomarkers. The first is the metabolites that accumulate due to the underlying defective lysosomal functions, for instances, the measurement of glucocerebroside in plasma of GD patients [16, 28]. Nevertheless, the elevations of these metabolic markers are usually not very pronounced and the normal levels are in relatively large ranges which make them not good biomarkers [28]. The second class is the plasma (or urinary) proteins that indirectly reflect the primary metabolic defects [16, 28]. For many years, abnormalities in serum levels of some macrophages-producing proteins such as tartrate-resisitant acid phosphatase (TRAP), hexosaminidase, angiotensin-converting enzyme (ACE) and lysozyme have been reported in GD patients [16, 28, 29]. However, they are not used as biomarkers for GD since their levels in GD patients may overlap to that of healthy individuals and they do not seem to be specific markers for Gaucher cells [28]. It was not until the early 1990s that the discovery of chitotriosidase (CT) has finally provided a surrogate marker for GD [28, 30]. CT is a human analogue of chitinases from lower organisms and is specifically produced by Gaucher cells but not normal tissue macrophages [28]. Serum from GD patients show a 1000 fold increase in CT level and CT level correlates well with glucocerebroside level in type 1 GD patients. Since glucocerebroside is the best quantitative measure for Gaucher cells, scientists have deduced that CT level is directly proportional to Gaucher cells mass [28]. Nonetheless, there are several limitations with the use of CT as biomarkers. Firstly, 6% of the general population lack CT activity as a result of mutation of the chitotriosidase gene [16, 30]. Moreover, the transglycosylation of the substrate, 4-methylumbelliferyl(MU)-chitotriose or 4MU-chitobiose, by the enzyme make measurement of the CT activity difficult as there is an apparent substrate inhibition effect [28,30]. Fortunately, the recent developed novel substrate, 4MU-deoxychitobiose has overcome this problem [28, 30]. Lastly, it is practically difficult to determine and interpret CT levels due to genetic variations [30]. A newly described chemokine, pulmonary and activation-regulated chemokine (PARC) also known as CCL18 has become particularly useful as a biomarker for patients who lack of CT [28, 29]. PARC levels of GD patients are increased by about 10-50 fold compare to healthy individuals with no overlapping values between the two groups. PARC is also produced and secreted by Gaucher cells but unlike CT it is detectable in all GD patients [29]. More recently, markedly elevations of serum levels of the chemokine, macrophage inflammatory protein (MIP)-1α and MIP-1β, are observed in GD patients. These proteins are not produced by Gaucher cells but the surrounding inflammatory cells [28]. A correlation between serum MIP-1β level and severity of the skeletal disease has been noticed, but yet more research is required before confirming the use of MIP-1β as a biomarker for GD [28].


Over the past decades, there were scattered cases reported the relationship between GD and Parkinson's disease (PD) which lead to recent larger researches in different ethics groups to evaluate the association between mutations of GCase and parkinsonism and the theory behind. Most of these studies have independently demonstrated an increase in GCase mutation in patients with sporadic PD.[31, 32, 33] An review summarised some of the major studies all over the world and concluded that the frequency of GCase mutations is increased around fivefold with PD. [31] According to research findings, hypothesis is made about the increase risk of developing dementia or cognitive impairment in early onset PD patients with GCase mutation.[32] The exact mechanism of the association between these two diseases remains unclear but several mechanisms has been proposed. Pathology of PD involved the aggregation of the insoluble protein alpha-synuclein which lead to the formation of Lewy body and it is suggested that GCase mutations could contribute to this aggregation. [31] GCase mutations usually involved missense alleles and lead to a misfold and unstable GCase which is degraded by the ubiqutin-proteasome system (UPS). Another hypothesis is that the unstable GCase might overwhelm the UPS and prevent degradation of the accumulated alpha-synuclein. [31, 32] One hypothesis is based on the fact that alpha-synuclein does bind with glycocerebroside or glycosphingolipids, and hence prevent the formation of fibrillar protein structures. [31] Lastly, it is suggested that glucocerebroside or ceramide metabolism could also contribute to the development of PD. [31,32] The objective of ongoing researches nowadays is to confirm the relationship between the two diseases and the mechanisms behind so as to determine whether other more promising therapies can be applied to PD patients with GCase mutations.[31]

LIMP-2 Receptor

The importance of mannose residues on GCase to target macrophages to improve internalization is discussed in the above passage. Once the exogenous GCase enter the macrophages, it needs to be targeted into the correct organelle, the lysosome, to elicit its clinical function. Unlike majority of lysosomal enzymes in which transport to lysosomes from the ER are mediated through M6P receptors, GCase is targeted to the lysosomes via a M6P-independent pathway. [34, 35] Lysosomal integral membrane protein type 2 (LIMP-2) is identified as the receptor responsible for the transport of GCase into lysosomes. [34, 35] In addition, studies also suggest that binding of newly synthesised GCase to LIMP-2 occurs in the ER rather than in the trans-Glogi network as in the M6P-mediated pathway. [34, 35]This result together with the observation that some of the GCase mutants, such as L444P, that retained in the ER normally can be transported to the lysosomes after coexpression of LIMP-2, suggest the therapeutic values of increasing LIMP-2 level in GD patients. [34]

Fig.8. Difference between sorting lysosomal hydrolase and GCase into lysosome via M6P receptor and LIMP-2 receptor respectively. Left: Lysosomal hydrolase interact with M6P receptor in the trans-Golgi network and M6P receptor is not present on lysosome. Right: Newly synthesized GCase interacts with LIMP-2 receptor on the ER. Dissociation between LIMP-2 and GCase occur when the complex reaches the lysosome.

The importance of LIMP-2 in GD is supported by researches showing a decrease in GCase level and activity in mouse tissue lacking LIMP-2. Since some mutations of the GCase can cause loss of interaction with the LIMP-2 leading to GD, it is important to identify specific GCase mutations that result in this loss of interactions and the role of this loss of interaction in pathologies, so that new pharmacological therapies can be developed.


Enzyme replacement therapy has a great impact on Gaucher Disease and other lysosomal storage disorders since its development and approval. Imiglucrease is proven to have an excellent efficacy and safety profile, however, its high cost in production has raise public concern of its cost effectiveness. The development of Velaglucerase alfa and prGCD is hoping to increase cost effectiveness of ERT by increasing clinical efficacy of the therapy and batch-to-batch reproducibility together with reduction of in vitro remodelling respectively. Apart from its cost, there are other limitations of ERT including ineffectiveness in neuropathic form of the disease and variation in response in different aspect of the disease. Other therapeutic options have been developed aiming to overcome the limitations of ERT. Substrate reduction therapy is based on preventing the accumulation of the substrate by partial inhibition in their synthesis. Miglustat is an inhibitor of the enzyme ceramide glucosyltransferase which is responsible for the biosynthesis of glycosphingolipids and is indicated for GD patients who do not tolerate ERT. [15,7,23] Chaperone mediated therapy is a newly derived therapeutic strategy which involve the use of small chemical chaperones to stabilize the correct folding of a protein and is undergoing clinical trials. [7, 14, 15, 23]Both of the new therapies involve smaller molecules so could be orally bioavailable and eliminate infusion-related reactions. [23] Furthermore, they have the potential to treat the neuropathic symptoms since they could cross the blood brain barrier and have different mechanism of actions. Gene therapy is the only curable therapy while all other therapies mentioned above require life-long treatments. [7] However, there are a number of hurdles needed to be overcome such as immune response, the use of viral vectors and the possibility of developing tumour. Up to date, many aspects related to GD remains unclear, for instance, the exact pathologies to neuropathic symptoms and skeletal disorders, the relationship with Parkinson's disease and Myeloma, identification of an accurate biomarker, mechanism of lysosomal trafficking and so on. Further investigations on all these fields are important for developing new pharmacological alternatives and better management of the disease All in all, the aim is to reduce the cost of treatment and improve the quality of life of the patient


Gaucher Disease, GD

Lysosomal Storage Disorder, LSD

Glucocerebrosidase; Acid-β-glucosidase, GCase

Central Nervous System, CNS

Endoplasmic Reticulum, ER

Endoplasmic Reticulum- Associated Degradation, ERAD

Enzyme Replacement Therapy, ERT

Food and Drug Administration, FDA

N-acetlyglucosamine, GlcNAC2

Mannose-6-Phosphate, M6P

International Collaborative Gaucher Group, ICGG

Mannose-Binding Lectin, MBL

Recombinant Plant-derived Glucocerebrosidase, prGCD

Tartrate-Resistant Acid Phosphatase, TRAP

Angiotensin-Converting Enzyme, ACE

Chitotriosidase, CT

Methylumbellifery, MU

Pulmonary and Activation-Regulated Chemokine, PARC

Macrophage Inflammatory Protein, MIP

Parkinson's disease, PD

Ubiqutin-Proteasome System, UPS

Lysosomal Integral Membrane Protein Type-2, LIMP-2