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Hudson H Freeze

Publications and source records attributed to Hudson H Freeze.

At least 19 recordsLinked to original sources
PubMed2006

Intracranial hemorrhage as the initial manifestation of a congenital disorder of glycosylation.

Intracranial hemorrhage in a term neonate is a rare event in the absence of an identifiable precipitating factor such as severe thrombocytopenia, mechanical trauma, asphyxia, infections, or congenital vascular malformations. Congenital disorders of glycosylation are a genetically and clinically heterogeneous group of multisystem disorders characterized by the abnormal glycosylation of a number of glycoproteins. Although bleeding caused by abnormal glycosylation of various coagulation factors is a well-known clinical complication of several types of congenital disorders of glycosylation, intracranial hemorrhage has not been reported as an initial manifestation of this entity. Here we report the detailed history of a family with 2 consecutive male infants, both born at term with intracranial hemorrhage diagnosed within the first 24 hours of life. The diagnosis of a congenital disorder of glycosylation was established in the second infant by an abnormal glycosylation of serum transferrin detected by electrospray-ionization mass spectrometry. Both infants showed significant neurologic deterioration during the first month of life, and both died at 5 months of age. Intracranial hemorrhage in a term neonate without a potential precipitating factor represents yet another clinical feature that should raise the suspicion for a congenital disorder of glycosylation.

Apnea↗
PubMed2006

Genetic defects in the human glycome.

The spectrum of all glycan structures--the glycome--is immense. In humans, its size is orders of magnitude greater than the number of proteins that are encoded by the genome, one percent of which encodes proteins that make, modify, localize or bind sugar chains, which are known as glycans. In the past decade, over 30 genetic diseases have been identified that alter glycan synthesis and structure, and ultimately the function of nearly all organ systems. Many of the causal mutations affect key biosynthetic enzymes, but more recent discoveries point to defects in chaperones and Golgi-trafficking complexes that impair several glycosylation pathways. As more glycosylation disorders and patients with these disorders are identified, the functions of the glycome are starting to be revealed.

Animals↗
PubMed2006

Heparan sulfate plays a central role in a dynamic in vitro model of protein-losing enteropathy.

Protein-losing enteropathy (PLE), the loss of plasma proteins through the intestine, is a symptom in ostensibly unrelated diseases. Emerging commonalities indicate that genetic insufficiencies predispose for PLE and environmental insults, e.g. viral infections and inflammation, trigger PLE onset. The specific loss of heparan sulfate (HS) from the basolateral surface of intestinal epithelial cells only during episodes of PLE suggests a possible mechanistic link. In the first tissue culture model of PLE using a monolayer of intestinal epithelial HT29 cells, we proved that HS loss directly causes protein leakage and amplifies the effects of the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha). Here, we extend our in vitro model to assess the individual and combined effects of HS loss, interferon gamma (IFNgamma), TNFalpha, and increased pressure, and find that HS plays a central role in the patho-mechanisms underlying PLE. Increased pressure, mimicking venous hypertension seen in post-Fontan PLE patients, substantially increased protein leakage, but HS loss, IFNgamma, or TNFalpha alone had only minor effects. However, IFNgamma up-regulated TNFR1 expression and amplified TNFalpha-induced protein leakage. IFNgamma and TNFalpha compromised the integrity of the HT29 monolayer and made it more susceptible to increased pressure. HS loss itself compromises the integrity of the monolayer, amplifying the effects of pressure, but also amplifies the effects of both cytokines. In the absence of HS a combination of increased pressure, IFNgamma, and TNFalpha caused maximum protein leakage. Soluble heparin fully compensated for HS loss, providing a reasonable explanation for patient favorable response to heparin therapy.

Albumins↗
PubMed2006

Congenital disorder of glycosylation Ic due to a de novo deletion and an hALG-6 mutation.

We describe a new cause of congenital disorder of glycosylation-Ic (CDG-Ic) in a young girl with a rather mild CDG phenotype. Her cells accumulated lipid-linked oligosaccharides lacking three glucose residues, and sequencing of the ALG6 gene showed what initially appeared to be a homozygous novel point mutation (338G>A). However, haplotype analysis showed that the patient does not carry any paternal DNA markers extending 33kb in the telomeric direction from the ALG6 region, and microsatellite analysis extended the abnormal region to at least 2.5Mb. We used high-resolution karyotyping to confirm a deletion (10-12Mb) [del(1)(p31.2p32.3)] and found no structural abnormalities in the father, suggesting a de novo event. Our findings extend the causes of CDG to larger DNA deletions and identify the first Japanese CDG-Ic mutation.

Carbohydrate Metabolism, Inborn Errors↗
PubMed2006

The congenital disorders of glycosylation: a multifaceted group of syndromes.

The congenital disorders of glycosylation (CDG) are a rapidly expanding group of metabolic syndromes with a wide symptomatology and severity. They all stem from deficient N-glycosylation of proteins. To date the group contains 18 different subtypes: 12 of Type I (disrupted synthesis of the lipid-linked oligosaccharide precursor) and 6 of Type II (malfunctioning trimming/processing of the protein-bound oligosaccharide). Main features of CDG involve psychomotor retardation; ataxia; seizures; retinopathy; liver fibrosis; coagulopathies; failure to thrive; dysmorphic features, including inverted nipples and subcutaneous fat pads; and strabismus. No treatment currently is available for the vast majority of these syndromes (CDG-Ib and CDG-IIc are exceptions), even though attempts to synthesize drugs for the most common subtype, CDG-Ia, have been made. In this review we will discuss the individual syndromes, with focus on their neuronal involvement, available and possible treatments, and future directions.

Carbohydrate Metabolism, Inborn Errors↗
PubMed2006

Cryptogenic liver disease in four children: a novel congenital disorder of glycosylation.

We investigated the metabolic defect(s) of four children who presented with isolated cryptogenic chronic liver disease, coagulopathy, and abnormalities of several unrelated serum glycoproteins. Analysis of the patients' serum glycoproteins and fibroblasts suggest they have a novel congenital disorder of glycosylation (CDG). All had abnormal transferrin (Tf) isoelectric focusing (IEF) profiles. More detailed analysis of Tf by electrospray ionization mass spectrometry (ESI-MS) showed a plethora of abnormal glycosylations that included loss of 1-2 sialic acids and 1-2 galactose units, typical of Group II defects. Tf from two patients also lacked 1-2 entire oligosaccharide chains, typical of Group One disorders. Total serum N-glycans were analyzed by HPLC and matrix-assisted laser desorption/ionization mass spectrometry and also showed increased proportion of neutral glycan chains lacking sialic acids and galactose units. Analysis of patient fibroblasts eliminated CDG-Ia, through CDG-Ih, -IL and CDG-IId. Our results suggest that a subset of children with clinically asymptomatic, cryptogenic hypertransaminasemia and/or liver steato-fibrosis may represent a novel type of CDG-X with an unknown defect(s). Clinicians are encouraged to test such patients for abnormal Tf glycosylation by ESI-MS.

Child, Preschool↗
PubMed2006

The Nuclear Overhauser Effect in the lead identification process.

In the past several years nuclear magnetic resonance (NMR) spectroscopy has emerged as a valuable tool in the drug discovery field. In such context, several NMR-based techniques have been developed aimed at the identification and subsequent optimization of novel binders for a given protein target. Among the different NMR approaches, those relying on the transferred Nuclear Overhauser Effect (tr-NOE) appear to be particularly useful as in some instances, in addition to binding, tr-NOE may provide also structural information on the binding mode of a ligand. In the current work we will reiterate the basic principles and applications that are related to measurements of tr-NOEs. The tr-NOE can be applied as a screening tool to recognize ligands for a given target protein in a mixture of compounds or to identify pair of molecules that bind to a protein simultaneously on adjacent sites (interligand NOEs). Moreover, in the case of peptide-ligands, tr-NOEs furnish intra-molecular distance constraints that can be used to determine their bioactive conformation. Starting from the conformation thus obtained, a pharmacophoric model can be derived and later used to search within a 3D database of small molecules to find new potentially active non-peptide compounds that fit the pharmacophore. We will report examples of each of the above mentioned strategies.

Magnetic Resonance Spectroscopy↗
PubMed2005

Applied glycoproteomics--approaches to study genetic-environmental collisions causing protein-losing enteropathy.

Protein-losing enteropathy (PLE), the loss of plasma proteins through the intestine, is a life-threatening symptom associated with seemingly unrelated conditions including Crohn's disease, congenital disorder of glycosylation, or Fontan surgery to correct univentricular hearts. Emerging commonalities between these and other disorders led us to hypothesize that PLE develops when genetic insufficiencies collide with simultaneous or sequential environmental insults. Most intriguing is the loss of heparan sulfate (HS) proteoglycans (HSPG) specifically from the basolateral surface of intestinal epithelial cells only during PLE episodes suggesting a direct link to protein leakage. Reasons for HSPG loss are unknown, but genetic insufficiencies affecting HSPG biosynthesis, trafficking, or degradation may be involved. Here, we describe cell-based assays we devised to identify key players contributing to protein leakage. Results from these assays confirm that HS loss directly causes protein leakage, but more importantly, it amplifies the effects of other factors, e.g., cytokines and increased pressure. Thus, HS loss appears to play a central role for PLE. To transfer our in vitro results back to the in vivo situation, we established methods to assess enteric protein leakage in mice and present several genetically deficient strains mimicking intestinal HS loss observed in PLE patients. Preliminary results indicate that mice with haploinsufficient genes involved in HS biosynthesis or HSPG trafficking develop intestinal protein leakage upon additional environmental stress. Our goal is to model PLE in vitro and in vivo to unravel the pathomechanisms underlying PLE, identify patients at risk, and provide them with a safe and effective therapy.

Animals↗
PubMed2005

Ablation of mouse phosphomannose isomerase (Mpi) causes mannose 6-phosphate accumulation, toxicity, and embryonic lethality.

MPI encodes phosphomannose isomerase, which interconverts fructose 6-phosphate and mannose 6-phosphate (Man-6-P), used for glycoconjugate biosynthesis. MPI mutations in humans impair protein glycosylation causing congenital disorder of glycosylation Ib (CDG-Ib), but oral mannose supplements normalize glycosylation. To establish a mannose-responsive mouse model for CDG-Ib, we ablated Mpi and provided dams with mannose to rescue the anticipated defective glycosylation. Surprisingly, although glycosylation was normal, Mpi(-/-) embryos died around E11.5. Mannose supplementation even hastened their death, suggesting that man-nose was toxic. Mpi(-/-) embryos showed growth retardation and placental hyperplasia. More than 90% of Mpi(-/-) embryos failed to form yolk sac vasculature, and 35% failed chorioallantoic fusion. We generated primary embryonic fibroblasts to investigate the mechanisms leading to embryonic lethality and found that mannose caused a concentration- and time-dependent accumulation of Man 6-P in Mpi(-/-) fibroblasts. In parallel, ATP decreased by more than 70% after 24 h compared with Mpi(+/+) controls. In cell lysates, Man-6-P inhibited hexokinase (70%), phosphoglucose isomerase (65%), and glucose-6-phosphate dehydrogenase (85%), but not phosphofructokinase. Incubating intact Mpi(-/-) fibroblasts with 2-[(3)H]deoxyglucose confirmed mannose-dependent hexokinase inhibition. Our results in vitro suggest that mannose toxicity in Mpi(-/-) embryos is caused by Man-6-P accumulation, which inhibits glucose metabolism and depletes intracellular ATP. This was confirmed in E10.5 Mpi(-/-) embryos where Man-6-P increased more than 10 times, and ATP decreased by 50% compared with Mpi(+/+) littermates. Because Mpi ablation is embryonic lethal, a murine CDG-Ib model will require hypomorphic Mpi alleles.

Adenosine Triphosphate↗
PubMed2005

Carboxylated glycans mediate colitis through activation of NF-kappa B.

The role of carbohydrate modifications of glycoproteins in leukocyte trafficking is well established, but less is known concerning how glycans influence pathogenesis of inflammation. We previously identified a carboxylate modification of N-linked glycans that is recognized by S100A8, S100A9, and S100A12. The glycans are expressed on macrophages and dendritic cells of normal colonic lamina propria, and in inflammatory infiltrates in colon tissues from Crohn's disease patients. We assessed the contribution of these glycans to the development of colitis induced by CD4(+)CD45RB(high) T cell transfer to Rag1(-/-) mice. Administration of an anti-carboxylate glycan Ab markedly reduced clinical and histological disease in preventive and early therapeutic protocols. Ab treatment reduced accumulation of CD4(+) T cells in colon. This was accompanied by reduction in inflammatory cells, reduced expression of proinflammatory cytokines and of S100A8, S100A9, and receptor for advanced glycation end products. In vitro, the Ab inhibited expression of LPS-elicited cytokines and induced apoptosis of activated macrophages. It specifically blocked activation of NF-kappaB p65 in lamina propria cells of colitic mice and in activated macrophages. These results indicate that carboxylate-glycan-dependent pathways contribute to the early onset of colitis.

Animals↗
PubMed2005

Clinical and molecular characterization of the first adult congenital disorder of glycosylation (CDG) type Ic patient.

Congenital disorder of glycosylation (CDG) type Ic, the second largest subtype of CDG, is caused by mutations in human ALG6 (hALG6). This gene encodes the alpha1,3-glucosyltransferase that catalyzes transfer of the first glucose residue to the lipid-linked oligosaccharide precursor for N-linked glycosylation. In this report, we describe the first adult patient diagnosed with CDG-Ic, carrying two previously unknown mutations. The first is a three base deletion (897-899delAAT) leading to the loss of I299, the second is an intronic mutation (IVS7 + 2T > G) that causes aberrant splicing. Wildtype hALG6, delivered by a lentiviral vector into patient's fibroblasts, clearly improves the biochemical phenotype, which confirms that the mutations are disease-causing. Striking clinical findings include limb deficiencies in the fingers, resembling brachydactyly type B, a deep vein thrombosis, pseudotumor cerebri, and endocrine disturbances with pronounced hyperandrogenism and virilization. However, even in adulthood, this patient shows normal magnetic resonance imaging of the brain.

Adult↗
PubMed2005

Hydrophobic Man-1-P derivatives correct abnormal glycosylation in Type I congenital disorder of glycosylation fibroblasts.

Patients with Type I congenital disorders of glycosylation (CDG-I) make incomplete lipid-linked oligosaccharides (LLO). These glycans are poorly transferred to proteins resulting in unoccupied glycosylation sequons. Mutations in phosphomannomutase (PMM2) cause CDG-Ia by reducing the activity of PMM, which converts mannose (Man)-6-P to Man-1-P before formation of GDP-Man. These patients have reduced Man-1-P and GDP-Man. To replenish intracellular Man-1-P pools in CDG-Ia cells, we synthesized two hydrophobic, membrane permeable acylated versions of Man-1-P and determined their ability to normalize LLO size and N-glycosylation in CDG-Ia fibroblasts. Both compounds, compound I (diacetoxymethyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate) (C-I) and compound II (diacetoxymethyl 2,3,4,6-tetra-O-ethyloxycarbonyl-alpha-D-mannopyranosyl phosphate) (C-II), contain two acetoxymethyl (CH2OAc) groups O-linked to phosphorous. C-I contains acetyl esters and C-II contains ethylcarbonate (CO2Et) esters on the Man residue. Both C-I and C-II normalized truncated LLO, but C-II was about 2-fold more efficient than C-I. C-II replenished the GDP-Man pool in CDG-Ia cells and was more efficiently incorporated into glycoproteins than exogenous Man at low concentrations (25-75 mM). In a glycosylation assay of DNaseI in CDG-Ia cells, C-II restored glycosylation to control cell levels. C-II also corrected impaired LLO biosynthesis in cells from a Dolichol (Dol)-P-Man deficient patient (CDG-Ie) and partially corrected LLO in cells from an ALG12 mannosyltransferase-deficient patient (CDG-Ig), whereas cells from an ALG3-deficient patient (CDG-Id) and from an MPDU1-deficient patient (CDG-If) were not corrected. These results validate the general concept of using pro-Man-1-P substrates as potential therapeutics for CDG-I patients.

Carbohydrate Conformation↗
PubMed2005

Novel carboxylated N-glycans contain oligosaccharide-linked glutamic acid.

We previously reported that N-glycans from bovine lung contain novel carboxylate groups. Here, we provide evidence that the carboxylated glycans contain glutamic acid. We labeled HeLa cells with [2,3-(3)H]glutamate and used a carboxylate-specific monoclonal antibody to enrich for the desired proteins. PNGaseF digestion of these proteins released labeled N-glycans with a free amino group and 1-3 carboxylates. Mild acid hydrolysis had no effect, but strong acid hydrolysis of the glycans released >80% of the (3)H as glutamate. Reducing the carboxylates to alcohols prior to hydrolysis eliminated the [(3)H]glutamate and generated [(3)H]4-amino 5-hydroxy pentanoic acid, suggesting that [(3)H]glutamate was linked to the glycan through its gamma-carboxyl. The glutamate-containing N-glycans resisted exoglycosidase digestion and oligosaccharide processing inhibitors greatly reduced [(3)H]glutamate incorporation. These results demonstrate that mammalian cells synthesize complex-type N-glycans with glutamate linked to their antennae, further expanding their potential for covalent or ionic interactions.

Acids↗
PubMed2005

Congenital disorder of glycosylation id presenting with hyperinsulinemic hypoglycemia and islet cell hyperplasia.

CONTEXT: Inborn errors in protein glycosylation, such as the congenital disorders of glycosylation (CDGs), generate multifaceted syndromes that impair many organ systems. We here report the diagnosis of the third known patient with CDG-Id. RESULTS: The patient's phenotype was extremely severe, and she succumbed at 19 d of age. Leading features included hyperinsulinemic hypoglycemia, and autopsy revealed islet cell hyperplasia with increased beta-cell mass. Other features were a Dandy-Walker malformation, facial dysmorphisms, and profound hypotonia. The patient carried a novel homozygous point mutation (512G>A) in the hALG3 gene, which encodes a mannosyltransferase. Lentiviral complementation with wild-type hALG3 corrects the biochemical defect in the patient's fibroblasts. CONCLUSIONS: Our findings underscore the importance of proper glycosylation in several major organ systems and emphasize that CDG should be ruled out in patients with persistent hyperinsulinemic hypoglycemia of unknown etiology.

Adult↗
PubMed2005

Altered glycan structures: the molecular basis of congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are a group of diseases that affect glycoprotein biogenesis. Eighteen different types of CDG have been defined genetically. They result from deficiencies in either the biosynthesis of oligosaccharide precursors or specific steps of N-glycan assembly, resulting in the absence or structural alteration of N-glycan chains. These diseases have a broad range of clinical phenotypes and affect nearly every organ system, with special emphasis on normal brain development and the multiple functions of the nervous, hepatic, gastrointestinal and immune systems. Although most of the deficiencies observed in CDG patients are only partial, the severity of the clinical manifestations signifies the relevance of protein N-glycosylation and shows the importance of defined glycan structures.

Carbohydrate Metabolism, Inborn Errors↗