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1.  Clinical utility of chitotriosidase enzyme activity in nephropathic cystinosis 
Nephropathic cystinosis is an inherited autosomal recessive lysosomal storage disorder characterized by the pathological accumulation and crystallization of cystine inside different cell types. WBC cystine determination forms the basis for the diagnosis and therapeutic monitoring with the cystine depleting drug (cysteamine). The chitotriosidase enzyme is a human chitinase, produced by activated macrophages. Its elevation is documented in several lysosomal storage disorders. Although, about 6% of Caucasians have enzyme deficiency due to homozygosity of 24-bp duplication mutation in the chitotriosidase gene, it is currently established as a screening marker and therapeutic monitor for Gaucher’s disease.
Plasma chitotriosidase activity was measured in 45 cystinotic patients, and compared with 87 healthy controls and 54 renal disease patients with different degrees of renal failure (CKD1-5). Chitotriosidase levels were also correlated with WBC cystine in 32 treated patients. Furthermore, we incubated control human macrophages in-vitro with different concentrations of cystine crystals and monitored the response of tumor necrosis factor-alpha (TNF-α) and chitotriosidase activity. We also compared plasma chitotriosidase activity in cystinotic knocked-out (n = 10) versus wild-type mice (n = 10).
Plasma chitotriosidase activity in cystinotic patients (0–3880, median 163 nmol/ml/h) was significantly elevated compared to healthy controls (0–90, median 18 nmol/ml/h) and to CKD patients (0–321, median 52 nmol/ml/h), P < 0.001 for both groups. Controls with decreased renal function had mild to moderate chitotriosidase elevations; however, their levels were significantly lower than in cystinotic patients with comparable degree of renal insufficiency. Chitotriosidase activity positively correlated with WBC cystine content for patients on cysteamine therapy (r = 0.8), P < 0.001. In culture, human control macrophages engulfed cystine crystals and released TNF-α into culture supernatant in a crystal concentration dependent manner. Chitotriosidase activity was also significantly increased in macrophage supernatant and cell-lysate. Furthermore, chitotriosidase activity was significantly higher in cystinotic knocked-out than in the wild-type mice, P = 0.003.
This study indicates that cystine crystals are potent activators of human macrophages and that chitotriosidase activity is a useful marker for this activation and a promising clinical biomarker and therapeutic monitor for nephropathic cystinosis.
PMCID: PMC4269071  PMID: 25407738
Lysosomal storage disorders; Nephropathic cystinosis; Cystine crystals; Macrophage activation; Chitotriosidase enzyme; Clinical screening; Cysteamine; Therapeutic monitoring
2.  Genetic defects in dolichol metabolism 
Congenital disorders of glycosylation (CDG) comprise a group of inborn errors of metabolism with abnormal glycosylation of proteins and lipids. Patients with defective protein N-glycosylation are identified in routine metabolic screening via analysis of serum transferrin glycosylation. Defects in the assembly of the dolichol linked Glc3Man9GlcNAc2 glycan and its transfer to proteins lead to the (partial) absence of complete glycans on proteins. These defects are called CDG-I and are located in the endoplasmic reticulum (ER) or cytoplasm. Defects in the subsequent processing of protein bound glycans result in the presence of truncated glycans on proteins. These defects are called CDG-II and the enzymes involved are located mainly in the Golgi apparatus. In recent years, human defects have been identified in dolichol biosynthesis genes within the group of CDG-I patients. This has increased interest in dolichol metabolism, has resulted in specific recognizable clinical symptoms in CDG-I and has offered new mechanistic insights in dolichol biosynthesis. We here review its biosynthetic pathways, the clinical and biochemical phenotypes in dolichol-related CDG defects, up to the formation of dolichyl-P-mannose (Dol-P-Man), and discuss existing evidence of regulatory networks in dolichol metabolism to provide an outlook on therapeutic strategies.
PMCID: PMC4281381  PMID: 25270028
3.  A compound heterozygous mutation in DPAGT1 results in a congenital disorder of glycosylation with a relatively mild phenotype 
Congenital disorders of glycosylation (CDG) are a large group of recessive multisystem disorders caused by impaired protein or lipid glycosylation. The CDG-I subgroup is characterized by protein N-glycosylation defects originating in the endoplasmic reticulum. The genetic defect is known for 17 different CDG-I subtypes. Patients in the few reported DPAGT1-CDG families exhibit severe intellectual disability (ID), epilepsy, microcephaly, severe hypotonia, facial dysmorphism and structural brain anomalies. In this study, we report a non-consanguineous family with two affected adults presenting with a relatively mild phenotype consisting of moderate ID, epilepsy, hypotonia, aggressive behavior and balance problems. Exome sequencing revealed a compound heterozygous missense mutation, c.85A>T (p.I29F) and c.503T>C (p.L168P), in the DPAGT1 gene. The affected amino acids are located in the first and fifth transmembrane domains of the protein. Isoelectric focusing and high-resolution mass spectrometry analyses of serum transferrin revealed glycosylation profiles that are consistent with a CDG-I defect. Our results show that the clinical spectrum of DPAGT1-CDG is much broader than appreciated so far.
PMCID: PMC3722673  PMID: 23249953
exome sequencing; intellectual disability; DPAGT1; congenital disorders of glycosylation; iso-electric focusing; mass spectrometry.
5.  Missense mutations in β-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) cause Walker–Warburg syndrome 
Human Molecular Genetics  2013;22(9):1746-1754.
Several known or putative glycosyltransferases are required for the synthesis of laminin-binding glycans on alpha-dystroglycan (αDG), including POMT1, POMT2, POMGnT1, LARGE, Fukutin, FKRP, ISPD and GTDC2. Mutations in these glycosyltransferase genes result in defective αDG glycosylation and reduced ligand binding by αDG causing a clinically heterogeneous group of congenital muscular dystrophies, commonly referred to as dystroglycanopathies. The most severe clinical form, Walker–Warburg syndrome (WWS), is characterized by congenital muscular dystrophy and severe neurological and ophthalmological defects. Here, we report two homozygous missense mutations in the β-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) gene in a family affected with WWS. Functional studies confirmed the pathogenicity of the mutations. First, expression of wild-type but not mutant B3GNT1 in human prostate cancer (PC3) cells led to increased levels of αDG glycosylation. Second, morpholino knockdown of the zebrafish b3gnt1 orthologue caused characteristic muscular defects and reduced αDG glycosylation. These functional studies identify an important role of B3GNT1 in the synthesis of the uncharacterized laminin-binding glycan of αDG and implicate B3GNT1 as a novel causative gene for WWS.
PMCID: PMC3613162  PMID: 23359570
6.  Heterozygosity for a Loss-of-Function Mutation in GALNT2 Improves Plasma Triglyceride Clearance in Man 
Cell metabolism  2011;14(6):811-818.
Genome-wide association studies have identified GALNT2 as a candidate gene in lipid metabolism, but it is not known how the encoded enzyme ppGal-NAc-T2, which contributes to the initiation of mucin-type O-linked glycosylation, mediates this effect. In two probands with elevated plasma high-density lipoprotein cholesterol and reduced triglycerides, we identified a mutation in GALNT2. It is shown that carriers have improved postprandial triglyceride clearance, which is likely attributable to attenuated glycosylation of apolipoprotein (apo) C-III, as observed in their plasma. This protein inhibits lipoprotein lipase (LPL), which hydrolyses plasma triglycerides. We show that an apoC-III-based peptide is a substrate for ppGalNAc-T2 while its glycosylation by the mutant enzyme is impaired. In addition, neuraminidase treatment of apoC-III which removes the sialic acids from its glycan chain decreases its potential to inhibit LPL. Combined, these data suggest that ppGalNAc-T2 can affect lipid metabolism through apoC-III glycosylation, thereby establishing GALNT2 as a lipid-modifying gene.
PMCID: PMC3523677  PMID: 22152306
8.  Mutations in ISPD cause Walker-Warburg syndrome and defective glycosylation of α-dystroglycan 
Nature genetics  2012;44(5):581-585.
Walker-Warburg syndrome (WWS) is an autosomal recessive multisystem disorder characterized by complex eye and brain abnormalities with congenital muscular dystrophy (CMD) and aberrant α-dystroglycan (αDG) glycosylation. Here, we report mutations in the isoprenoid synthase domain-containing (ISPD) gene as the second most common cause of WWS. Bacterial IspD is a nucleotidyl transferase belonging to a large glycosyltransferase family, but its role in chordates has been obscure to date because this phylum does not have the corresponding non-mevalonate isoprenoid biosynthesis pathway. Knockdown of ispd in zebrafish recapitulates the human WWS phenotype with hydrocephalus, reduced eye size, muscle degeneration and hypoglycosylated αDG. These results implicate a role for ISPD in αDG glycosylation to maintain sarcolemma integrity in vertebrates.
PMCID: PMC3378661  PMID: 22522421
9.  From discrete dilated cardiomyopathy to successful cardiac transplantation in congenital disorders of glycosylation due to dolichol kinase deficiency (DK1-CDG) 
Heart Failure Reviews  2012;18(2):187-196.
Congenital disorders of glycosylation are a growing group of inborn errors of protein glycosylation. Cardiac involvement is frequently observed in the most common form, PMM2-CDG, especially hypertrophic cardiomyopathy. Dilated cardiomyopathy, however, has been only observed in a few CDG subtypes, usually with a lethal outcome. We report on cardiac pathology in nine patients from three unrelated Israeli families, diagnosed with dolichol kinase deficiency, due to novel, homozygous DK1 gene mutations. The cardiac symptoms varied from discrete, mild dilation to overt heart failure with death. Two children died unexpectedly with acute symptoms of heart failure before the diagnosis of DK1-CDG and heart transplantation could take place. Three other affected children with mild dilated cardiomyopathy at the time of the diagnosis deteriorated rapidly, two of them within days after an acute infection. They all went through successful heart transplantation; one died unexpectedly and 2 others are currently (after 1–5 years) clinically stable. The other 4 children diagnosed with mild dilated cardiomyopathy are doing well on supportive heart failure therapy. In most cases, the cardiac findings dominated the clinical picture, without central nervous system or multisystem involvement, which is unique in CDG syndrome. We suggest to test for DK1-CDG in patients with dilated cardiomyopathy. Patients with discrete cardiomyopathy may remain stable on supportive treatment while others deteriorate rapidly. Our paper is the first comprehensive study on the phenotype of DK1-CDG and the first successful organ transplantation in CDG syndrome.
PMCID: PMC3593007  PMID: 22327749
Dilated cardiomyopathy; Heart failure; CDG-Im; Congenital disorders of glycosylation; Dolichol kinase deficiency; Cardiac transplantation
10.  Autosomal Recessive Dilated Cardiomyopathy due to DOLK Mutations Results from Abnormal Dystroglycan O-Mannosylation 
PLoS Genetics  2011;7(12):e1002427.
Genetic causes for autosomal recessive forms of dilated cardiomyopathy (DCM) are only rarely identified, although they are thought to contribute considerably to sudden cardiac death and heart failure, especially in young children. Here, we describe 11 young patients (5–13 years) with a predominant presentation of dilated cardiomyopathy (DCM). Metabolic investigations showed deficient protein N-glycosylation, leading to a diagnosis of Congenital Disorders of Glycosylation (CDG). Homozygosity mapping in the consanguineous families showed a locus with two known genes in the N-glycosylation pathway. In all individuals, pathogenic mutations were identified in DOLK, encoding the dolichol kinase responsible for formation of dolichol-phosphate. Enzyme analysis in patients' fibroblasts confirmed a dolichol kinase deficiency in all families. In comparison with the generally multisystem presentation in CDG, the nonsyndromic DCM in several individuals was remarkable. Investigation of other dolichol-phosphate dependent glycosylation pathways in biopsied heart tissue indicated reduced O-mannosylation of alpha-dystroglycan with concomitant functional loss of its laminin-binding capacity, which has been linked to DCM. We thus identified a combined deficiency of protein N-glycosylation and alpha-dystroglycan O-mannosylation in patients with nonsyndromic DCM due to autosomal recessive DOLK mutations.
Author Summary
Idiopathic dilated cardiomyopathy (DCM) is estimated to be of genetic origin in 20%–48% of the patients. Almost all currently known genetic defects show dominant inheritance, although especially in younger children recessive causes have been proposed to contribute considerably to DCM. Knowledge of the genetic causes and pathophysiological mechanisms is essential for prognosis and treatment. Here, we studied several individual young patients (5–13 years old) with idiopathic and sometimes asymptomatic dilated cardiomyopathy. The key to identification of the gene was the finding of abnormal protein N-glycosylation. Via homozygosity mapping and functional knowledge of the N-glycosylation pathway, the causative gene could be identified as dolichol kinase (DOLK). Since DCM is very rare in N-glycosylation disorders (Congenital Disorders of Glycosylation, CDG) and most patients with CDG present with a multisystem involvement, we studied the underlying pathophysiological cause of this life-threatening disease. Biochemical experiments in affected heart tissue showed deficient O-mannosylation of alpha-dystroglycan, which could be correlated with the dilated cardiomyopathy. Our results thus highlight nonsyndromic DCM as a novel presentation of DOLK-CDG, via deficient O-mannosylation of alpha-dystroglycan.
PMCID: PMC3248466  PMID: 22242004
11.  How to find and diagnose a CDG due to defective N-glycosylation 
PMCID: PMC3137781  PMID: 21739167
12.  Reply to Leao-Teles et al 
PMCID: PMC2987312
13.  Transferrin mutations at the glycosylation site complicate diagnosis of congenital disorders of glycosylation type I 
Congenital disorders of glycosylation (CDG) form a group of metabolic disorders caused by deficient glycosylation of proteins and/or lipids. Isoelectric focusing (IEF) of serum transferrin is the most common screening method to detect abnormalities of protein N-glycosylation. On the basis of the IEF profile, patients can be grouped into CDG type I or CDG type II. Several protein variants of transferrin are known that result in a shift in isoelectric point (pI). In some cases, these protein variants co-migrate with transferrin glycoforms, which complicates interpretation. In two patients with abnormal serum transferrin IEF profiles, neuraminidase digestion and subsequent IEF showed profiles suggestive of the diagnosis of CDG type I. Mass spectrometry of tryptic peptides of immunopurified transferrin, however, revealed a novel mutation at the N-glycan attachment site. In case 1, a peptide with mutation p.Asn630Thr in the 2nd glycosylation site was identified, resulting in an additional band at disialotransferrin position on IEF. After neuraminidase digestion, a single band was found at the asialotransferrin position, indistinguishable from CDG type I patients. In case 2, a peptide with mutation p.Asn432His was found. These results show the use of mass spectrometry of transferrin peptides in the diagnostic track of CDG type I.
PMCID: PMC3137782  PMID: 21431619
14.  From glycosylation disorders to dolichol biosynthesis defects: a new class of metabolic diseases 
Polyisoprenoid alcohols are membrane lipids that are present in every cell, conserved from archaea to higher eukaryotes. The most common form, alpha-saturated polyprenol or dolichol is present in all tissues and most organelle membranes of eukaryotic cells. Dolichol has a well defined role as a lipid carrier for the glycan precursor in the early stages of N-linked protein glycosylation, which is assembled in the endoplasmic reticulum of all eukaryotic cells. Other glycosylation processes including C- and O-mannosylation, GPI-anchor biosynthesis and O-glucosylation also depend on dolichol biosynthesis via the availability of dolichol-P-mannose and dolichol-P-glucose in the ER. The ubiquity of dolichol in cellular compartments that are not involved in glycosylation raises the possibility of additional functions independent of these protein post-translational modifications. The molecular basis of several steps involved in the synthesis and the recycling of dolichol and its derivatives is still unknown, which hampers further research into this direction. In this review, we summarize the current knowledge on structural and functional aspects of dolichol metabolites. We will describe the metabolic disorders with a defect in known steps of dolichol biosynthesis and recycling in human and discuss their pathogenic mechanisms. Exploration of the developmental, cellular and biochemical defects associated with these disorders will provide a better understanding of the functions of this lipid class in human.
PMCID: PMC3137772  PMID: 21384228
15.  Readily Accessible Bicyclononynes for Bioorthogonal Labeling and Three-Dimensional Imaging of Living Cells** 
PMCID: PMC3021724  PMID: 20857472
cycloadditions; cycloalkynes; imaging; kinetics; protein modification
16.  SRD5A3 is required for the conversion of polyprenol to dolichol, essential for N-linked protein glycosylation 
Cell  2010;142(2):203-217.
N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the Congenital Disorders of Glycosylation (CDG). We describe a new type of CDG caused by mutations in the steroid 5α-reductase type 3 (SRD5A3) gene. Patients have mental retardation, ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation.
PMCID: PMC2940322  PMID: 20637498
N-glycosylation; dolichol; polyprenol; SRD5A3
17.  Loss-of-function mutations in ATP6V0A2 impair vesicular trafficking, tropoelastin secretion and cell survival 
Human Molecular Genetics  2009;18(12):2149-2165.
Autosomal recessive cutis laxa type 2 (ARCL2), a syndrome of growth and developmental delay and redundant, inelastic skin, is caused by mutations in the a2 subunit of the vesicular ATPase H+-pump (ATP6V0A2). The goal of this study was to define the disease mechanisms that lead to connective tissue lesions in ARCL2. In a new cohort of 17 patients, DNA sequencing of ATP6V0A2 detected either homozygous or compound heterozygous mutations. Considerable allelic and phenotypic heterogeneity was observed, with a missense mutation of a moderately conserved residue p.P87L leading to unusually mild disease. Abnormal N- and/or mucin type O-glycosylation was observed in all patients tested. Premature stop codon mutations led to decreased ATP6V0A2 mRNA levels by destabilizing the mutant mRNA via the nonsense-mediated decay pathway. Loss of ATP6V0A2 either by siRNA knockdown or in ARCL2 cells resulted in distended Golgi cisternae, accumulation of abnormal lysosomes and multivesicular bodies. Immunostaining of ARCL2 cells showed the accumulation of tropoelastin (TE) in the Golgi and in large, abnormal intracellular and extracellular aggregates. Pulse–chase studies confirmed impaired secretion and increased intracellular retention of TE, and insoluble elastin assays showed significantly reduced extracellular deposition of mature elastin. Fibrillin-1 microfibril assembly and secreted lysyl oxidase activity were normal in ARCL2 cells. TUNEL staining demonstrated increased rates of apoptosis in ARCL2 cell cultures. We conclude that loss-of-function mutations in ATP6V0A2 lead to TE aggregation in the Golgi, impaired clearance of TE aggregates and increased apoptosis of elastogenic cells.
PMCID: PMC2685755  PMID: 19321599
18.  Autosomal recessive cutis laxa syndrome revisited 
European Journal of Human Genetics  2009;17(9):1099-1110.
The clinical spectrum of the autosomal recessive cutis laxa syndromes is highly heterogeneous with respect to organ involvement and severity. One of the major diagnostic criteria is to detect abnormal elastin fibers. In several other clinically similar autosomal recessive syndromes, however, the classic histological anomalies are absent, and the definite diagnosis remains uncertain. In cutis laxa patients mutations have been demonstrated in elastin or fibulin genes, but in the majority of patients the underlying genetic etiology remains unknown. Recently, we found mutations in the ATP6V0A2 gene in families with autosomal recessive cutis laxa. This genetic defect is associated with abnormal glycosylation leading to a distinct combined disorder of the biosynthesis of N- and O-linked glycans. Interestingly, similar mutations have been found in patients with wrinkly skin syndrome, without the presence of severe skin symptoms of elastin deficiency. These findings suggest that the cutis laxa and wrinkly skin syndromes are phenotypic variants of the same disorder. Interestingly many phenotypically similar patients carry no mutations in the ATP6V0A2 gene. The variable presence of protein glycosylation abnormalities in the diverse clinical forms of the wrinkled skin-cutis laxa syndrome spectrum necessitates revisiting the diagnostic criteria to be able to offer adequate prognosis assessment and counseling. This paper aims at describing the spectrum of clinical features of the various forms of autosomal recessive cutis laxa syndromes. Based on the recently unraveled novel genetic entity we also review the genetic aspects in cutis laxa syndromes including genotype–phenotype correlations and suggest a practical diagnostic approach.
PMCID: PMC2986595  PMID: 19401719
cutis laxa; wrinkled skin; congenital disorders of glycosylation; gerodermia osteodysplastica; De Barsy Syndrome; ATP6V0A2
19.  Th1-Directing Adjuvants Increase the Immunogenicity of Oligosaccharide-Protein Conjugate Vaccines Related to Streptococcus pneumoniae Type 3 
Infection and Immunity  2003;71(12):6915-6920.
Oligosaccharide (OS)-protein conjugates are promising candidate vaccinesagainst encapsulated bacteria, such as Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae. Although the effects of several variables such as OS chain length and protein carrier have been studied, little is known about the influence of adjuvants on the immunogenicity of OS-protein conjugates. In this study, a minimal protective trisaccharide epitope of Streptococcus pneumoniae type 3 conjugated to the cross-reacting material of diphtheria toxin was used for immunization of BALB/c mice in the presence of different adjuvants. Subsequently, half of the mice received a booster immunization with conjugate alone. Independent of the use and type of adjuvant, all mice produced long-lasting anti-polysaccharide type 3 (PS3) antibody levels, which provided full protection against challenge with pneumococcal type 3 bacteria. All adjuvants tested increased the anti-PS3 antibody levels and opsonic capacities as measured by an enzyme-linked immunosorbent assay and an in vitro phagocytosis assay. The use of QuilA or a combination of the adjuvants CpG and dimethyl dioctadecyl ammonium bromide resulted in the highest phagocytic capacities and the highest levels of Th1-related immunoglobulin G (IgG) subclasses. Phagocytic capacity correlated strongly with Th1-associated IgG2a and IgG2b levels, to a lesser extent with Th2-associated IgG1 levels, and weakly with thiocyanate elution as a measure of avidity. Thus, the improved immunogenicity of OS-protein conjugates was most pronounced for Th1-directing adjuvants.
PMCID: PMC308892  PMID: 14638780
20.  Synthetic Polysaccharide Type 3-Related Di-, Tri-, and Tetrasaccharide–CRM197 Conjugates Induce Protection against Streptococcus pneumoniae Type 3 in Mice 
Infection and Immunity  2001;69(7):4698-4701.
Di-, tri-, and tetrasaccharides, synthesized according to the chemical structure of pneumococcal polysaccharide type 3 (PS3), were coupled to the cross-reactive material (CRM197) of modified diphtheria toxin in different molar carbohydrate/protein ratios using the squarate coupling method. To study protective immunity, female BALB/c mice were subcutaneously immunized twice (with a 3-week interval) using the amount of conjugates corresponding to 2.5 μg of oligosaccharide per mouse. The conjugates evoked PS3 binding immunoglobulin G antibodies that lasted for at least 7 weeks after the booster. Immunogenicity was not influenced by the carbohydrate/protein ratio. All mice with PS3-specific antibodies survived the intraperitoneal challenge with Streptococcus pneumoniae type 3. Therefore, synthetic oligosaccharide-protein conjugates might have potential as vaccines.
PMCID: PMC98553  PMID: 11402020

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