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.     

Core fucosylation of N-linked glycans in leukocyte adhesion deficiency/congenital disorder of glycosylation IIc fibroblasts

Leukocyte adhesion deficiency/congenital disorder of glycosylation IIc (LAD II/CDG IIc) is a genetic disease characterized by a decreased expression of fucose in glycoconjugates, resulting in leukocyte adhesion deficiency and severe morphological and neurological abnormalities. The biochemical defect is a reduced transport of guanosine diphosphate-L-fucose (GDP-L-fucose) from cytosol into the Golgi compartment, which reduces its availability as substrate for fucosyltransferases. The aim of this study was to determine the effects of a limited supply of GDP-L-fucose inside the Golgi on core fucosylation (alpha1,6-fucose linked to core N-acetylglucosamine [GlcNAc]) of N-linked glycans in LAD II fibroblasts. The results showed that, although [3H]fucose incorporation was generally reduced in LAD II cells, core fucosylation was affected to a greater extent compared with other types of fucosylation of N-linked oligosaccharides. In particular, core fucosylation was found to be nearly absent in biantennary negatively charged oligosaccharides, whereas other types of structures, in particular triantennary neutral species, were less affected by the reduction. Expression and activity of alpha1,6-fucosyltransferase (FUT8) in control and LAD II fibroblasts were comparable, thus excluding the possibility of a decreased activity of the transferase. The data obtained confirm that the concentration of GDP-L-fucose inside the Golgi can differentially affect the various types of fucosylation in vivo and also indicate that core fucosylation is not dependent only on the availability of GDP-L-fucose, but it is significantly influenced by the type of oligosaccharide structure. The relevant reduction in core fucosylation observed in some species of oligosaccharides could also provide clues for the identification of glycans involved in the severe developmental abnormalities observed in LAD II.     

Mass spectrometric approach for screening modifications of total serum N-glycome in human diseases: application to cirrhosis

Congenital and acquired modifications of glycosylation in diseases are a rapidly growing field that demonstrates the importance of glycosylation in human biology. Unfortunately, in clinical biochemistry, very few tests are available to explore oligosaccharide metabolism on a large scale. Such an assay needs to be of high throughput, rapid, and preferentially noninvasive. In the present study, we describe a method to analyze qualitative variations of N-glycosylation of human serum proteins. The method is based on direct release of N-linked oligosaccharides from patient serum samples, a single-step purification, and a matrix-assisted laser desorption ionization time of flight mass spectrometric analysis. A complementary structural study of the released oligosaccharides was achieved by enzymatic digestions, linkage analysis, and electrospray ionization ion trap mass spectrometry (ESI-IT-MS) of the permethylated N-glycome. A total of 26 oligosaccharide structures were individualized, their presence in human serum being the result of the combination of the biosynthesis and catabolic pathways. Application of the protocol to the serum of patients with cirrhosis demonstrates the ability of this assay to identify acquired modifications of glycosylation. Furthermore, we have analyzed the N-glycans and showed the increase in bisecting N-acetylglucosamine residue, core fucosylation, and the presence of an important population of neutral oligosaccharides. The study of total serum N-glycome modifications is a preliminary for the discovery of new noninvasive diagnostic or prognostic biomarkers resulting from the variations of the N-glycan metabolism during diseases.     

Serum N-glycan and O-glycan analysis by mass spectrometry for diagnosis of congenital disorders of glycosylation

Congenital disorders of glycosylation (CDGs) are caused by defects in genes that participate in biosynthetic glycosylation pathways. To date, 19 different genetic defects in N-glycosylation, 17 in O-glycosylation, and 21 in multiple glycosylation are known. Current diagnostic testing of CDGs largely relies on indirect analysis of glycosylation of serum transferrin. Such analysis alone is insufficient to diagnose many of the known glycosylation disorders. To improve the diagnosis of these groups of CDGs, we have developed serum or plasma N- and O-glycan profiling using a combination of MALDI–TOF/MS and LC–MS/MS technologies. Using this approach, we analyzed samples from nine patients with different known multiple glycosylation disorders, including three with COG deficiencies, one with TMEM165-CDG, two with PGM1-CDG, and three with SLC35A2-CDG, and one patient with combined type I and type II of unknown molecular etiology. Measurement of the relative quantities of various N- and O-glycan species clearly differentiates patients and controls. Our study demonstrates that structural analysis and quantitation of combined N- and O-glycan profiles are reliable diagnostic tools for CDGs.     

Structural characterization of the N-glycan moiety and site of glycosylation in vitellogenin from the decapod crustacean Cherax quadricarinatus

Glycosylation is of importance for the structure and function of proteins. In the case of vitellin (Vt), a ubiquitous protein accumulated into granules as the main yolk protein constituent of oocytes during oogenesis, glycosylation could be of importantance for the folding, processing and transport of the protein to the yolk and also provides a source of carbohydrate during embryogenesis. Vt from the crayfish Cherax quadricarinatus is synthesized as a precursor protein, vitellogenin (Vg), in the hepatopancreas, transferred to the hemolymph, and mobilized into the growing oocyte via receptor-mediated endocytosis. The gene sequence of C. quadricarinatus shows a 2584-amino-acid protein with 10 putative glycosylation sites. In this study a combined approach of lectin immunoblotting, in-gel deglycosylation, and mass spectrometry was used to identify the glycosylation sites and probe the structure of the glycan moieties using C. quadricarinatus Vg as a model system. Three of the consensus sites for N-glycosylation-namely, Asn(152), Asn(160) and Asn(2493)-were glycosylated with the high-mannose glycans, Man(5-9)GlcNAc(2), and the glucose-capped oligosaccharide Glc(1)Man(9)GlcNAc(2).     

Hypoglycosylation with increased fucosylation and branching of serum transferrin N-glycans in untreated galactosemia

Untreated classic galactosemia (galactose-1-phosphate uridyltransferase [GALT] deficiency) is known as a secondary congenital disorders of glycosylation (CDG) characterized by galactose deficiency of glycoproteins and glycolipids (processing defect or CDG-II). The mechanism of this undergalactosylation has not been established. Here we show that in untreated galactosemia, there is also a partial deficiency of whole glycans of serum transferrin associated with increased fucosylation and branching as seen in genetic glycosylation assembly defects (CDG-I). Thus galactosemia seems to be a secondary "dual" CDG causing a processing as well as an assembly N-glycosylation defect. We also demonstrated that in galactosemia patients, transferrin N-glycan biosynthesis is restored upon dietary treatment.     

Identification of phosphorylated oligosaccharides in cells of patients with a congenital disorders of glycosylation (CDG-I)

Protein N-glycosylation is initiated by the dolichol cycle in which the oligosaccharide precursor Glc₃Man₉GlcNAc₂-PP-dolichol is assembled in the endoplasmic reticulum (ER). One critical step in the dolichol cycle concerns the availability of Dol-P at the cytosolic face of the ER membrane. In RFT1 cells, the lipid-linked oligosaccharide (LLO) intermediate Man₅GlcNAc₂-PP-Dol accumulates at the cytosolic face of the ER membrane. Since Dol-P is a rate-limiting intermediate during protein N-glycosylation, continuous accumulation of Man₅GlcNAc₂-PP-Dol would block the dolichol cycle. Hence, we investigated the molecular mechanisms by which accumulating Man₅GlcNAc₂-PP-Dol could be catabolized in RFT1 cells. On the basis of metabolic labeling experiments and in comparison to human control cells, we identified phosphorylated oligosaccharides (POS), not found in human control cells and present evidence that they originate from the accumulating LLO intermediates. In addition, POS were also detected in other CDG patients’ cells accumulating specific LLO intermediates at different cellular locations. Moreover, the enzymatic activity that hydrolyses oligosaccharide-PP-Dol into POS was identified in human microsomal membranes and required Mn²⁺ for optimal activity. In CDG patients’ cells, we thus identified and characterized POS that could result from the catabolism of accumulating LLO intermediates.     

Identification of glycoconjugates in the urine of a patient with congenital disorder of glycosylation by high-resolution mass spectrometry

More than 150 molecular species were detected in a single glycoconjugate fraction obtained from urine of a congenital disorders of glycosylation (CDG) patient by use of high-resolution FT-ICR MS. With respect to its high-mass accuracy and resolving power, FT-ICR MS represents an ideal tool for analysis of single components in complex glycoconjugate mixtures obtained from body fluids. The presence of overlapping nearly isobaric ionic species in glycoconjugate mixtures obtained from CDG patient's urine was postulated from fragmentation data of several precursor ions obtained by nanoESI Q-TOF CID. Their existence was confirmed by high-resolution/high-mass accuracy FT-ICR MS detection. High-resolution FT-ICR mass spectra can, therefore, be generally considered for glycoscreening of complex mixture samples in a single stage. From the accurate molecular ion mass determinations the composition of glycoconjugate species can be identified. Particular enhancement of identification is offered by computer-assisted calculations in combination with monosaccharide building block analysis, which can be extended by considerations of non-carbohydrate modifications, such as amino acids, phosphates and sulfates. Taking advantage of this strategy, the number of compositions assigned to mass peaks was significantly increased in a fraction obtained from urine by size exclusion and anion exchange chromatography.     

Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis

The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDGs). In these diseases, many biological functions are compromised, giving rise to a wide range of severe clinical conditions. By performing detailed analyses of the total serum glycoproteins as well as isolated transferrin and IgG, we have directly correlated aberrant glycosylation with a faulty glycosylation processing step. In one patient the complete absence of complex type sugars was consistent with ablation of GlcNAcTase II activity. In another CDG type II patient, the identification of specific hybrid sugars suggested that the defective processing step was cell type-specific and involved the mannosidase III pathway. In each case, complementary serum proteome analyses revealed significant changes in some 31 glycoproteins, including components of the complement system. This biochemical approach to charting diseases that involve alterations in glycan processing provides a rapid indicator of the nature, severity, and cell type specificity of the suboptimal glycan processing steps; allows links to genetic mutations; indicates the expression levels of proteins; and gives insight into the pathways affected in the disease process.     

Increased alpha3-fucosylation of alpha(1)-acid glycoprotein in patients with congenital disorder of glycosylation type IA (CDG-Ia)

Increased fucosylation of the type (sialyl) Lewis(x) was detected on the acute-phase plasma protein alpha(1)-acid glycoprotein (AGP) in patients with the congenital disorder of glycosylation type IA. This is remarkable, because in these patients the biosynthesis of guanosine 5'-diphosphate (GDP)-D-mannose is strongly decreased, and GDP-D-mannose is the direct precursor for GDP-L-fucose, the substrate for fucosyltransferases. The concomitantly occurring increased branching of the glycans of AGP and the increased fucosyltransferase activity in plasma suggest that a chronic hepatic inflammatory reaction has induced the increase in fucosylation.     

Phosphomannomutase activity in congenital disorders of glycosylation type Ia determined by direct analysis of the interconversion of mannose-1-phosphate to mannose-6-phosphate by high-pH anion-exchange chromatography with pulsed amperometric detection

Congenital disorders of glycosylation (CDG) are a group of multisystemic disorders resulting from defects in the synthesis and processing of N-linked oligosaccharides. The most common form, CDG type Ia (CDG-Ia), results from a deficiency of the enzyme phosphomannomutase (PMM). PMM converts mannose 6-phosphate (man-6-P) to mannose-1-phosphate (man-1-P), which is required for the synthesis of GDP-mannose, a substrate for dolichol-linked oligosaccharide synthesis. The traditional assay for PMM, a coupled enzyme system based on the reduction of NADP(+) to NADPH using man-1-P as a substrate, has limitations in accuracy and reproducibility. Therefore, a more sensitive, direct test for PMM activity, based on the detection of the conversion of man-1-P to man-6-P by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), was developed. Using this assay, the activity of PMM was markedly deficient in fibroblasts and lymphoblasts from 23 patients with CDG-Ia (range 0-15.3% of control, average 4.9+/-4.7%) and also decreased in seven obligate heterozygotes (range 33.0-72.0% of control, average 52.2+/-14.7%). Unlike the spectrophotometric method, there was no overlap in PMM activity among patients, obligate heterozygotes, or controls. Thus, the PMM assay based on HPAEC-PAD has increased utility in the clinical setting, and can be used, together with transferrin isoelectric focusing, to diagnose patients with CDG-Ia and to identify heterozygotes when clinically indicated.     

Engineering novel Lec1 glycosylation mutants in CHO-DUKX cells: molecular insights and effector modulation of N-acetylglucosaminyltransferase I

Many secreted or cell surface proteins are post-translationally modified by carbohydrate chains which are a primary source of heterogeneity. The Lec1 mutant, which is defective in Golgi N-acetylglucosaminyltransferase I (GnTI) activity, produces relatively homogeneous Man(5) GlcNAc(2) glycan modifications, and is widely used for various applications. To facilitate the investigation of GnTI, its Man5 glycan endproduct, and the impact of Man5 on effector function, the present study has established several novel Lec1 mutants in dhfr(-) CHO-DUKX cells through chemical mutagenesis and lectin selection. A total of nine clonal lines exhibiting the Lec1-phenotype are characterized, six of which harbor non-sense mutations leading to a truncated GnTI, and three (R415K, D291N, and P138L) of which are novel loss-of-function sense mutations. Analysis of the rabbit GnTI structure (Unligil et al., 2000) indicates that D291 is the proposed catalytic base and R415 is a crucial residue in forming the substrate binding pocket, whereas P138 is key to maintaining two β strands in proximity to the substrate binding pocket. Computational modeling reveals that the oligomannose glycan backbone of a glycoprotein (the acceptor substrate) fits nicely into the unoccupied channel of the substrate binding pocket partly through hydrogen bonding with R415 and D291. This finding is consistent with the ordered sequential Bi Bi kinetic mechanism suggested for GnTI, in which binding of UDP-GlcNAc (the donor substrate)/Mn(2+) induces conformational changes that promote acceptor binding. When an anti-human CD20 antibody protein is stably expressed in one CHO-DUKX-Lec1 line, it is confirmed that N-glycans are predominantly Man(5) GlcNAc(2) and they do not contain an α1,6-fucose linked to the innermost GlcNAc. Furthermore, this Man(5) GlcNAc(2) modified antibody exhibits a significantly increased ADCC activity than the wild-type protein, while displaying a lower CDC activity. The data support the hypothesis that modulating GnTI activity can influence antibody effector functions for proteins with an IgG1 immunoglobulin Fc domain.     

II. Model Building: An Electrical Theory of Control of Growth and Development in Animals,Prompted by Studies of Exogenous Magnetic Field Effects (Paper I), and Evidence of DNA Current Conduction,In Vitro

A theory of control of cellular proliferation and differentiation in the early development of metazoan systems, postulating a system of electrical controls “parallel” to the processes of molecular biochemistry, is presented. It is argued that the processes of molecular biochemistry alone cannot explain how a developing organism defies a stochastic universe.

The demonstration of current flow (charge transfer) along the long axis of DNA through the base-pairs (the “π-way) in vitro raises the question of whether nature may employ such current flows for biological purposes. Such currents might be too small to be accessible to direct measurement in vivo but conduction has been measured in vitro, and the methods might well be extended to living systems. This has not been done because there is no reasonable model which could stimulate experimentation. We suggest several related, but detachable or independent, models for the biological utility of charge transfer, whose scope admittedly outruns current concepts of thinking about organization, growth, and development in eukaryotic, metazoan systems. The ideas are related to explanations proposed to explain the effects demonstrated on tumors and normal tissues described in Article I (this issue).

Microscopic and mesoscopic potential fields and currents are well known at sub-cellular, cellular, and organ systems levels. Not only are such phenomena associated with internal cellular membranes in bioenergetics and information flow, but remarkable long-range fields over tissue interfaces and organs appear to play a role in embryonic development (Nuccitelli, ). The origin of the fields remains unclear and is the subject of active investigation. We are proposing that similar processes could play a vital role at a “sub-microscopic level,” at the level of the chromosomes themselves, and could play a role in organizing and directing fundamental processes of growth and development, in parallel with the more discernible fields and currents described.     

Role of the conserved oligomeric Golgi (COG) complex in protein glycosylation

The Golgi apparatus is a central hub for both protein and lipid trafficking/sorting and is also a major site for glycosylation in the cell. This organelle employs a cohort of peripheral membrane proteins and protein complexes to keep its structural and functional organization. The conserved oligomeric Golgi (COG) complex is an evolutionary conserved peripheral membrane protein complex that is proposed to act as a retrograde vesicle tethering factor in intra-Golgi trafficking. The COG protein complex consists of eight subunits, distributed in two lobes, Lobe A (Cog1-4) and Lobe B (Cog5-8). Malfunctions in the COG complex have a significant impact on processes such as protein sorting, glycosylation, and Golgi integrity. A deletion of Lobe A COG subunits in yeasts causes severe growth defects while mutations in COG1, COG7, and COG8 in humans cause novel types of congenital disorders of glycosylation. These pathologies involve a change in structural Golgi phenotype and function. Recent results indicate that down-regulation of COG function results in the resident Golgi glycosyltransferases/glycosidases to be mislocalized or degraded.     

Existence of both kappa and lambda light chain messenger RNA sequences in mouse myeloma, MOPC-104E, known as a lambda chain producer.

A mouse myeloma, MOPC-104E, which is known to synthesize and secrete λ type light chain protein as a constituent of immunoglobulin M, was shown to contain mRNA sequences coding for κ as well as λ type light chain protein. Light chain mRNA sequences were quantitated by nucleic acid hybridization reaction using radioactive DNA complementary to light chain mRNAs which had been purified from other myelomas. The amount of κ type light chain mRNA present in MOPC-104E is almost equivalent to that of λ type light chain mRNA. κ chain mRNA was not separated from λ chain mRNA either by centrifugation in sucrose density gradient or by polyacrylamide gel electrophoresis in formamide.     

Evolution of MHC class I genes in two ancient fish, paddlefish (Polyodon spathula) and Chinese sturgeon (Acipenser sinensis)

Here we present the first isolation of major histocompatibility complex (MHC) class I genes from two ancient fish, paddlefish (Polyodon spathula) and Chinese sturgeon (Acipenser sinensis). Seventeen sequences obtained showed high polymorphism and positive natural selection with d_N/d_S > 1. Evolutionary relationships revealed that sequences from paddlefish and Chinese sturgeon distinguished from other vertebrate class I and had an intermingling of alleles, which indicates that Acipenseriformes have a common ancestral gene of class I and a trans-species polymorphism across Acipenseriformes. We also found clear evidence of recombination among class I genes of paddlefish and Chinese sturgeon.