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.     

Golgi GDP-fucose transporter-deficient mice mimic congenital disorder of glycosylation IIc/leukocyte adhesion deficiency II

Modification of glycoproteins by the attachment of fucose residues is widely distributed in nature. The importance of fucosylation has recently been underlined by identification of the monogenetic inherited human disease "congenital disorder of glycosylation IIc," also termed "leukocyte adhesion deficiency II." Due to defective Golgi GDP-fucose transporter (SLC35C1) activity, patients show a hypofucosylation of glycoproteins and present clinically with mental and growth retardation, persistent leukocytosis, and severe infections. To investigate effects induced by the loss of fucosylated structures in different organs, we generated a mouse model for the disease by inactivating the Golgi GDP-transporter gene (Slc35c1). Lectin binding studies revealed a tremendous reduction of fucosylated glycoconjugates in tissues and isolated cells from Slc35c1(-/-) mice. Fucose treatment of cells from different organs led to partial normalization of the fucosylation state of glycoproteins, thereby indicating an alternative GDP-fucose transport mechanism. Slc35c1-deficient mice presented with severe growth retardation, elevated postnatal mortality rate, dilatation of lung alveoles, and hypocellular lymph nodes. In vitro and in vivo leukocyte adhesion and rolling assays revealed a severe impairment of P-, E-, and L-selectin ligand function. The diversity of these phenotypic aspects demonstrates the broad general impact of fucosylation in the mammalian organism.     

Contribution of N-linked glycans to the conformation and function of intercellular adhesion molecules (ICAMs)

The crystal structures of the glycosylated N-terminal two domains of ICAM-1 and ICAM-2 provided a framework for understanding the role of glycosylation in the structure and function of intercellular adhesion molecules (ICAMs). The most conserved glycans were less flexible in the structures, interacting with protein residues and contributing to receptor folding and expression. The first N-linked glycan in ICAM-2 contacts an exposed tryptophan residue, defining a conserved glycan-W motif critical for the conformation of the integrin binding domain. The absence of this motif in human ICAM-1 exposes regions used in receptor dimerization and rhinovirus recognition. Experiments with soluble molecules having the N-terminal two domains of human ICAMs identified glycans of the high mannose type N-linked to the second domain of the dendritic cell-specific ICAM-grabbing nonintegrin lectin-ligands ICAM-2 and ICAM-3. About 40% of those receptor molecules bear endoglycosidase H sensitive glycans responsible of the lectin binding activity. High mannose glycans were absent in ICAM-1, which did not bind to the lectin, but they appeared in ICAM-1 mutants with additional N-linked glycosylation and lectin binding activity. N-Linked glycosylation regulate both conformation and immune related functions of ICAM receptors.     

Integrin-dependent leukocyte adhesion involves geranylgeranylated protein(s)

Integrin-dependent leukocyte adhesion is modulated by alterations in receptor affinity or by post-receptor events. Pretreatment of Jurkat T-cells with the 3-hydroxymethylglutaryl-coenzyme A reductase inhibitor, lovastatin, markedly reduced (IC(50) approximately 1-2 microM) alpha(4)beta(1)-dependent adhesion to fibronectin (FN) stimulated by phorbol 12-myristate 13-acetate (PMA) which modulates post-receptor events. In contrast, lovastatin did not inhibit Jurkat cell adhesion to FN induced by the beta(1) integrin-activating monoclonal antibody (mAb) 8A2, which directly modulates beta(1) integrin affinity. Similarly, pretreatment of U937 cells with lovastatin inhibited PMA-stimulated, but not mAb 8A2-stimulated, alpha(6)beta(1)-dependent leukocyte adhesion to laminin. The inhibition of lovastatin on PMA-stimulated leukocyte adhesion was not mediated by mitogen-activated protein kinase or phosphatidylinositol 3-kinase pathway. The inhibitory effect of lovastatin on PMA-stimulated leukocyte adhesion was reversed by co-incubation with geranylgeraniol, but not with farnesol, with concurrent reversal of the inhibition of protein prenylation as shown by protein RhoA geranylgeranylation. The selective inhibition of protein geranylgeranylation by the specific protein geranylgeranyltransferase-I inhibitor, GGTI-298, blocked PMA-stimulated leukocyte adhesion but not mAb 8A2-induced leukocyte adhesion. The protein farnesyltransferase inhibitor, FTI-277, had no effect on leukocyte adhesion induced by either stimulus. These results demonstrate that protein geranylgeranylation, but not farnesylation, is required for integrin-dependent post-receptor events in leukocyte adhesion.     

Hypoglycosylation of a brain glycoprotein ({beta}-trace protein) in CDG syndromes due to phosphomannomutase deficiency and N-acetylglucosaminyl-transferase II deficiency

Human β-trace protein is a major intrathecally synthesizedpolypeptide constituent of human cerebrospinal fluid. We havepreviously shown that this protein is almost quantitativelymodified with biantennary complex-type N-linked oli-gosaccharideswhich show "brain-type" glycosylation characteristics (Hoffmann,A.et al, J. Neurochenu, 63, pp. 2185-2191,1994). In the presentstudy human β-trace protein from the cerebrospinal fluid(CSF) of patients with carbohydrate-deficient glycoprotein syndrome(CDGS) due to phospho-mannomutase (PMM) deficiency and N-acetyl-glucosami-nyltransferaseII (GlcNAc-T II) deficiency as well as from control individualswas studied by Western blot analysis. The protein from pooledCSFs was purified by immunoaffinity chromatography. The proteinfrom the five patients with CDGS PMM deficiency showed threeprotein bands upon SDS-PAGE analysis corresponding to the di-,mono-, and unglycosylated polypeptide forms. Carbohydrate structuralanalysis of the enzymatically liberated N-glycans was performedapplying mapping by HPAEC-PAD, methylation analysis as wellas MALD/TOF-MS. Essentially identical oli-gosaccharide structureswere detected in β-TP from type I patients and controladult pooled CSF. The β-trace protein from two patientswith GlcNAc-T II deficiency showed a single di-N-glycosylatedprotein band with a significantly lower molecular weight thanthe di-glycosylated polypeptide from control patients and theβ-trace protein from pooled adult CSF. β-TP from GlcNAc-TII deficiency patients shared only three oligosaccharides outof the 13 observed in β-TP from controls or patients withPMM deficiency. The major oligosaccharide structures of theglycoprotein from patients with GlcNAc-T n deficiency were foundto be monoanten-nary asialo- or monosialylated lactosamine-typechains with proximal fucose and bisecting GlcNAc. "brain-type" N-glycosylation carbohydrate deficiency glycoprotein syndrome human β-trace protein phosphomannomutase deficiency GlcNAc-transferase II deficiency human cerebrospinal fluid     

Detection of the causal mutation for canine leukocyte adhesion deficiency (CLAD) using pyrosequencing

A missense mutation in the ITGB2 gene causes canine leukocyte adhesion deficiency (CLAD) in Irish setters. We constructed a diagnostic test to identify heterozygous CLAD carriers based on a newly developed technology termed pyrosequencing. Although primarily designed for high-speed generation of DNA sequence in a gel-free system, the technology can be applied to rapid single-nucleotide polymorphism analysis in a clinical setting. The testing of 339 dogs originating from a total of 10 countries was conducted and CLAD carriers were identified within every country where more than one sample was analysed. This indicates that the CLAD mutation is widespread and that there is a strong need for a robust diagnostic test.     

A novel leukocyte adhesion deficiency III variant: kindlin-3 deficiency results in integrin- and nonintegrin-related defects in different steps of leukocyte adhesion

Leukocyte adhesion deficiency type III is a recently described condition involving a Glanzmann-type bleeding syndrome and leukocyte adhesion deficiency. This was ascribed to a defect of the FERMT3 gene resulting in abnormal expression of kindlin-3, a protein expressed in hematopoietic cells with a major role in the regulation of integrin activation. In this article, we describe a patient with a new mutation of FERMT3 and lack of kindlin-3 expression in platelets and leukocytes. We assayed quantitatively the first steps of kindlin-3-defective leukocyte adhesion, namely, initial bond formation, bond strengthening, and early spreading. Initial bond formation was readily stimulated with neutrophils stimulated by fMLF, and neutrophils and lymphocytes stimulated by a phorbol ester or Mn(2+). In contrast, attachment strengthening was defective in the patient's lymphocytes treated with PMA or Mn(2+), or fMLF-stimulated neutrophils. However, attachment strengthening was normal in patient's neutrophils treated with phorbol ester or Mn(2+). In addition, the patient's T lymphocytes displayed defective integrin-mediated spreading and a moderate but significant decrease of spreading on anti-CD3-coated surfaces. Patient's neutrophils displayed a drastic alteration of integrin-mediated spreading after fMLF or PMA stimulation, whereas signaling-independent Mn(2+) allowed significant spreading. In conclusion, the consequences of kindlin-3 deficiency on β(2) integrin function depend on both cell type and the stimulus used for integrin activation. Our results suggest looking for a possible kindlin-3 involvement in membrane dynamical event independent of integrin-mediated adhesion.     

Low incidence of bovine leukocyte adhesion deficiency (BLAD) carriers in Indian cattle and buffalo breeds

BLAD is an autosomal recessive genetic disease that affects Holstein-Friesian (HF) cattle worldwide. It is a disease characterized by a reduced expression of the adhesion molecules on neutrophils. The disease is caused by a mutation that replaces adenine at 383 with guanine, which causes an amino acid change from aspartic acid to glycine. Blood samples and a few semen samples were collected from 1250 phenotypically normal individuals, including HF (N=377), HF crossbred (N=334), Jersey (105), other breeds of cattle (N=160) and water buffalo Bubalus bubalis (N=274) belonging to various artificial insemination stations, bull mother farms (BMFs) and embryo transfer (ET) centres across the country. PCR-RFLP was performed to detect a point mutation in CD18, surface molecules of neutrophils. The results indicate that out of 1250 cattle and buffaloes tested for BLAD, 13 HF purebreds out of 377 and 10 HF crossbreds out of 334 appear to be BLAD carriers. In the HF and HF crossbred population, the percentage of BLAD carriers was estimated as 3.23%. The condition is alarming as the mutant gene has already entered the HF crossbred cattle population and therefore, the population of HF and its crossbreds needs regular screening to avoid the risk of spreading BLAD in the breeding cattle population of India.     

Role of N-linked glycans of envelope glycoproteins in infectivity of human immunodeficiency virus type 1.

We have shown that enzymatic removal of N-linked glycans from human immunodeficiency virus type 1 (HIV-1) recombinant envelope glycoproteins gp160 and gp120 produced in BHK-21 cells did not significantly reduce their ability to bind to CD4, the cellular receptor for the virus. Because recombinant proteins may behave differently from proteins present on virions, we investigated whether such viral envelope glycoproteins either in a purified form or present on viral particles could be deglycosylated by treatment with an endoglycosidase F-N-glycanase mixture which cleaves all accessible glycan moieties. Endoglycosidase analysis of the carbohydrate composition of purified viral gp120 (vgp120) indicated a glycosylation pattern similar to that for recombinant gp120 (rgp120), and treatment with endoglycosidase F-N-glycanase resulted in comparable molecular weight (MW) reduction for both molecules. Similarly, after immunoblotting of the deglycosylated viral preparation, the characteristic 160- and 120-kilodalton (kDa) bands were replaced by 90- and 60-kDa bands, respectively. The apparent MW of gp41 shifted to 35 kDa. These results are consistent with complete deglycosylation. The immunoreactive conformation of envelope glycoproteins remained unaltered after deglycosylation: they were recognized to the same extent by specific human polyclonal or mouse monoclonal antibodies, and no proteolysis of viral proteins occurred during enzymatic treatment. Deglycosylation of vgp120 resulted in a less than 10-fold reduction of the ability to bind to CD4, presented either in a soluble form or at the cell membrane. In addition, deglycosylation significantly reduced, but did not abolish, HIV-1 binding to and infectivity of CD4+ cells as determined, respectively, by an indirect immunofluorescence assay and a quantitative dose-response infection assay. Taken together, these results indicate that removal of glycans present on mature envelope glycoproteins of HIV-1 diminishes but does not abolish either virus binding to CD4 or its capacity to infect CD4+ cells.     

Two N-linked glycans are required to maintain the transport activity of the bile salt export pump (ABCB11) in MDCK II cells

The aim of this study was to determine the role of N-linked glycosylation in protein stability, intracellular trafficking, and bile acid transport activity of the bile salt export pump [Bsep (ATP-binding cassette B11)]. Rat Bsep was fused with yellow fluorescent protein, and the following mutants, in which Asn residues of putative glycosylation sites (Asn(109), Asn(116), Asn(122), and Asn(125)) were sequentially replaced with Gln, were constructed by site-directed mutagenesis: single N109Q, double N109Q + N116Q, triple N109Q + N116Q + N122Q, and quadruple N109Q + N116Q + N122Q + N125Q. Immunoblot and glycosidase cleavage analysis demonstrated that each site was glycosylated. Removal of glycans decreased taurocholate transport activity as determined in polarized MDCK II cells. This decrease resulted from rapid decay of the mutant Bsep protein; biochemical half-lives were 3.76, 3.65, 3.24, 1.35, and 0.52 h in wild-type, single-mutant, double-mutant, triple-mutant, and quadruple-mutant cells, respectively. Wild-type and single- and double-mutant proteins were distributed exclusively along the apical membranes, whereas triple- and quadruple-mutant proteins remained intracellular. MG-132 but not bafilomycin A(1) extended the half-life, suggesting a role for the proteasome in Bsep degradation. To determine whether a specific glycosylation site or the number of glycans was critical for protein stability, we studied the protein expression of combinations of N-glycan-deficient mutants and observed that Bsep with one glycan was considerably unstable compared with Bsep harboring two or more glycans. In conclusion, at least two N-linked glycans are required for Bsep protein stability, intracellular trafficking, and function in the apical membrane.     

Sulfated N-linked oligosaccharides in mammalian cells. II. Identification of glycosaminoglycan-like chains attached to complex-type glycans

In the preceding paper (Roux, L., Holojda, S., Sundblad, G., Freeze, H. H., and Varki, A. (1988) J. Biol. Chem. 263, 8879-8889) we described the metabolic labeling and isolation of sulfated N-linked oligosaccharides from mammalian cell lines. All cell lines studied contained a class of sulfated sialylated complex-type chains with 2-6 negative charges. In this paper, we show that bovine pulmonary arterial endothelial (CPAE) and human erythroleukemia (K562) cell lines also contain a class of more highly charged sulfated but less sialylated oligosaccharides. These molecules were further characterized by ion exchange chromatography and various enzymatic and chemical treatments. In both cell lines they contained greater than 6 negative charges, but those from K562 were even more highly charged than those from CPAE. Nitrous acid, heparinase, and heparitinase degradation of K562 oligosaccharides released 88, 64, and 78%, respectively, of 35S label. Combined digestion with the two enzymes resulted in 87% release. The corresponding values for CPAE were 48, 25, and 50% (60% for the two enzymes together). Chondroitinase ABC (or AC) digestion of K562 and CPAE oligosaccharides released 10 and 5%, respectively. About 30% of the 35S-labeled oligosaccharides from CPAE were sensitive to endo-beta-galactosidase, indicating that poly-N-acetyl-lactosamine structures were present on some chains. Highly charged [3H]mannose-labeled sulfated oligosaccharides from CPAE cells became neutral after treatment with heparinase/heparitinase but were resistant to Pronase, further proving that glycosaminoglycan (GAG)-like chains were directly attached to N-linked oligosaccharides. Such neutralized oligosaccharides did not bind to concanavalin A-Sepharose, but some interacted with phytohemagglutinin L4, indicating that they were bi-, tri-, or tetra-antennary complex-type chains. Thus, K562 and CPAE cells contain different types of GAG chains directly attached to asparagine-linked oligosaccharides. Such molecules were not found in many other cell lines that synthesize the more typical O-linked GAG chains. This suggests that the occurrence of these novel N-linked chains is not a random event resulting from accidental initiation of GAG chain synthesis on N-linked intermediates in the Golgi apparatus.     

A missense mutation in the beta-2 integrin gene (ITGB2) causes canine leukocyte adhesion deficiency.

Canine leukocyte adhesion deficiency (CLAD) is a fatal immunodeficiency disease found in Irish setters. The clinical manifestations of CLAD are very similar to LAD in humans and BLAD in cattle, which are both caused by mutations in ITGB2 encoding the leukocyte integrin beta-2 subunit (CD18). Sequence analysis of the ITGB2 coding sequence from a CLAD dog and a healthy control revealed a single missense mutation, Cys36Ser. This cysteine residue is conserved among all beta integrins, and the mutation most likely disrupts a disulfide bond. The mutation showed a complete association with CLAD in Irish setters and was not found in a sample of dogs from other breeds. The causative nature of this mutation was confirmed by transduction experiments using retroviral vectors and human LAD EBV B-cells. The normal canine CD18 formed heterodimers with the human CD11 subunit, whereas gene transfer of the mutant CD18 resulted in very low levels of CD11/CD18 expression. The identification of the causative mutation for CLAD now makes it possible to identify carrier animals with a simple diagnostic DNA test, and it forms the basis for using CLAD as a large animal model for the development and evaluation of clinical treatments for human LAD.     

Unusual expression of IgG Fc receptors on peripheral granulocytes from patients with leukocyte adhesion deficiency (CD11/CD18 deficiency)

Leukocyte adhesion deficiency (LAD) is a hereditary disease characterized by defective expression of leukocyte adhesion glycoproteins; lymphocyte function-associated Ag-1 (CD11a/CD18), CR3 (CD11b/CD18) and p150,95 (CD11c/CD18). Granulocytes, monocytes, and lymphocytes of patients with LAD show profoundly defective in vivo and in vitro adherence-dependent immune functions. We investigated the expression of FcR for IgG on polymorphonuclear cells (PMN) and monocytes from patients with LAD, and their luminol- and lucigenin-enhanced chemiluminescence production in response to SRBC sensitized with murine (m) IgG2a and IgG2b. Unstimulated patient PMN showed an enhanced chemiluminescence in response to mIgG2a-SRBC and an increased phagocytosis of mIgG2a-SRBC. The up-regulated functions were inhibited by monomeric human IgG in a dose-dependent manner, which was attributed to an increase in expression of FcRI on patient PMN, as shown by flow cytometry using monoclonal antibody, 32.2, specific for human FcRI. In contrast, neither the expression of FcR on the monocytes of LAD patients nor their FcR-mediated functions were different from those of controls.     

Role of N-linked glycans on bunyamwera virus glycoproteins in intracellular trafficking, protein folding, and virus infectivity

The membrane glycoproteins (Gn and Gc) of Bunyamwera virus (BUN, family Bunyaviridae) contain three potential sites for the attachment of N-linked glycans: one site (N60) on Gn and two (N624 and N1169) on Gc. We determined that all three sites are glycosylated. Digestion of the glycoproteins with endo-beta-N-acetylglucosaminidase H (endo H) or peptide:N-glycosidase F revealed that Gn and Gc differ significantly in their glycan status and that late in infection Gc glycans remain endo H sensitive. The roles of the N-glycans in intracellular trafficking of the glycoproteins to the Golgi, protein folding, and virus replication were investigated by mutational analysis and confocal immunofluorescence. Elimination of the glycan on Gn, by changing N60 to a Q residue, resulted in the protein misfolding and failure of both Gn and Gc proteins to traffic to the Golgi complex. We were unable to rescue a viable virus by reverse genetics from a cDNA containing the N60Q mutation. In contrast, mutant Gc proteins lacking glycans on either N624 or N1169, or both sites, were able to target to the Golgi. Gc proteins containing mutations N624Q and N1169Q acquired endo H resistance. Three viable N glycosylation-site-deficient viruses, lacking glycans on one site or both sites on Gc, were created by reverse genetics. The viability of these recombinant viruses and analysis of growth kinetics indicates that the glycans on Gc are not essential for BUN replication, but they do contribute to the efficiency of virus infection.     

A novel 3' splice-site mutation and a novel gross deletion in leukocyte adhesion deficiency (LAD)-1

A patient was diagnosed with leukocyte adhesion deficiency-1. She was born in 1996 and her parents are not known to be related. Her leukocytes expressed less than 2% of the CD18 antigens relative to normal individuals. Molecular analysis revealed that she is a compound heterozygote. She inherited a 27,703 bp deletion from her father (g.43201_PTTG1IP:10890del27703), spanning from intron 11 of the gene for the β2 integrin (ITGB2, CD18, NG_007270.2) to intron 2 of the gene for the Pituitary Tumor-Transforming Gene 1 Interacting Protein (PTTG1IP, NC_000021.8). The maternal allele has a g.23457C>A mutation at position −10 in intron 2 of the ITGB2 gene, resulting in the activation of a cryptic 3′ splice site in intron 2 to include 43 intronic nucleotides (r.[59-43_59-1ins;59-10C>A]).