A Novel Type of Macrothrombocytopenia Associated with a Defect in α2,3-Sialylation
2011; Elsevier BV; Volume: 179; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2011.06.012
ISSN1525-2191
AutoresClaire Jones, Jonas Denecke, Ronald Sträter, Torsten Stölting, Yvonne Schunicht, Dagmar Zeuschner, Judith Klumperman, Dirk J. Lefeber, Oliver Spelten, Alexander Zarbock, Sørge Kelm, Karen Strenge, Stuart M. Haslam, Kerstin Lühn, Dorothea Stahl, Luca Gentile, Thomas Schreiter, Philip Hilgard, Annette G. Beck‐Sickinger, Thorsten Marquardt, Martin K. Wild,
Tópico(s)Platelet Disorders and Treatments
ResumoWe describe a novel type of human thrombocytopenia characterized by the appearance of giant platelets and variable neutropenia. Searching for the molecular defect, we found that neutrophils had strongly reduced sialyl-Lewis X and increased Lewis X surface expression, pointing to a deficiency in sialylation. We show that the glycosylation defect is restricted to α2,3-sialylation and can be detected in platelets, neutrophils, and monocytes. Platelets exhibited a distorted structure of the open canalicular system, indicating defective platelet generation. Importantly, patient platelets, but not normal platelets, bound to the asialoglycoprotein receptor (ASGP-R), a liver cell-surface protein that removes desialylated thrombocytes from the circulation in mice. Taken together, this is the first type of human thrombocytopenia in which a specific defect of α2,3-sialylation and an induction of platelet binding to the liver ASGP-R could be detected. We describe a novel type of human thrombocytopenia characterized by the appearance of giant platelets and variable neutropenia. Searching for the molecular defect, we found that neutrophils had strongly reduced sialyl-Lewis X and increased Lewis X surface expression, pointing to a deficiency in sialylation. We show that the glycosylation defect is restricted to α2,3-sialylation and can be detected in platelets, neutrophils, and monocytes. Platelets exhibited a distorted structure of the open canalicular system, indicating defective platelet generation. Importantly, patient platelets, but not normal platelets, bound to the asialoglycoprotein receptor (ASGP-R), a liver cell-surface protein that removes desialylated thrombocytes from the circulation in mice. Taken together, this is the first type of human thrombocytopenia in which a specific defect of α2,3-sialylation and an induction of platelet binding to the liver ASGP-R could be detected. Several types of hereditary macrothrombocytopenia have been described, including Bernard-Soulier syndrome and the May-Hegglin anomaly, which are caused by mutations in the genes coding for platelet glycoproteins (GP) Ib/IX and nonmuscle myosin heavy chain, respectively.1Kunishima S. Kamiya T. Saito H. Genetic abnormalities of Bernard-Soulier syndrome.Int J Hematol. 2002; 76: 319-327Crossref PubMed Scopus (75) Google Scholar, 2Seri M. Cusano R. Gangarossa S. Caridi G. Bordo D. Lo Nigro C. Ghiggeri G.M. Ravazzolo R. Savino M. Del Vecchio M. D'Apolito M. Iolascon A. Zelante L.L. Savoia A. Balduini C.L. Noris P. Magrini U. Belletti S. Heath K.E. Babcock M. Glucksman M.J. Aliprandis E. Bizzaro N. Desnick R.J. Martignetti J.A. Mutations in MYH9 result in the May-Hegglin anomaly, and Fechtner and Sebastian syndromes The May-Heggllin/Fechtner Syndrome Consortium.Nat Genet. 2000; 26: 103-105Crossref PubMed Scopus (370) Google Scholar Macrothrombocytopenia can also be caused by a defect in glycosylation.3Willig T.B. Breton-Gorius J. Elbim C. Mignotte V. Kaplan C. Mollicone R. Pasquier C. Filipe A. Mielot F. Cartron J.P. Gougerot-Pocidalo M.A. Debili N. Guichard J. Dommergues J.P. Mohandas N. Tchernia G. Macrothrombocytopenia with abnormal demarcation membranes in megakaryocytes and neutropenia with a complete lack of sialyl-Lewis-X antigen in leukocytes–a new syndrome?.Blood. 2001; 97: 826-828Crossref PubMed Scopus (39) Google Scholar This disease, termed congenital disorder of glycosylation-IIf (CDG-IIf), was detected in a child whose neutrophils lacked expression of the sialic acid–containing tetrasaccharide sialyl-Lewis X (sLex) and showed increased expression of its nonsialylated form Lewis X (Lex). Notably, abnormal demarcation membranes in megakaryocytes strongly pointed to a defect in the generation of thrombocytes.3Willig T.B. Breton-Gorius J. Elbim C. Mignotte V. Kaplan C. Mollicone R. Pasquier C. Filipe A. Mielot F. Cartron J.P. Gougerot-Pocidalo M.A. Debili N. Guichard J. Dommergues J.P. Mohandas N. Tchernia G. Macrothrombocytopenia with abnormal demarcation membranes in megakaryocytes and neutropenia with a complete lack of sialyl-Lewis-X antigen in leukocytes–a new syndrome?.Blood. 2001; 97: 826-828Crossref PubMed Scopus (39) Google Scholar Mutations in the gene encoding the Golgi transporter for cytidine monophosphate (CMP)-sialic acid were described as the cause for this disease.4Martinez-Duncker I. Dupre T. Piller V. Piller F. Candelier J.J. Trichet C. Tchernia G. Oriol R. Mollicone R. Genetic complementation reveals a novel human congenital disorder of glycosylation of type II, due to inactivation of the Golgi CMP-sialic acid transporter.Blood. 2005; 105: 2671-2676Crossref PubMed Scopus (126) Google Scholar Generally, sialylation appears to strongly affect the number of circulating platelets. Indeed, desialylation of platelets results in their clearance from the circulation.5Greenberg J. Packham M.A. Cazenave J.P. Reimers H.J. Mustard J.F. Effects on platelet function of removal of platelet sialic acid by neuraminidase.Lab Invest. 1975; 32: 476-484PubMed Google Scholar Moreover, mice deficient for α2,3-sialyltransferase IV show strong thrombocytopenia.6Ellies L.G. Ditto D. Levy G.G. Wahrenbrock M. Ginsburg D. Varki A. Le D.T. Marth J.D. Sialyltransferase ST3Gal-IV operates as a dominant modifier of hemostasis by concealing asialoglycoprotein receptor ligands.Proc Natl Acad Sci U S A. 2002; 99: 10042-10047Crossref PubMed Scopus (163) Google Scholar, 7Ellies L.G. Sperandio M. Underhill G.H. Yousif J. Smith M. Priatel J.J. Kansas G.S. Ley K. Marth J.D. Sialyltransferase specificity in selectin ligand formation.Blood. 2002; 100: 3618-3625Crossref PubMed Scopus (123) Google Scholar Interestingly, as in CDG-IIf, platelets are enlarged in these mice. Recently, experiments in mice have shown that hyposialylated platelets readily bind to the liver asialoglycoprotein receptor (ASGP-R) in vivo and are efficiently removed from the circulation by the ASGP-R, explaining the induction of thrombocytopenia.8Grewal P.K. Uchiyama S. Ditto D. Varki N. Le D.T. Nizet V. Marth J.D. The Ashwell receptor mitigates the lethal coagulopathy of sepsis.Nat Med. 2008; 14: 648-655Crossref PubMed Scopus (280) Google Scholar, 9Sorensen A.L. Rumjantseva V. Nayeb-Hashemi S. Clausen H. Hartwig J.H. Wandall H.H. Hoffmeister K.M. Role of sialic acid for platelet life span: exposure of beta-galactose results in the rapid clearance of platelets from the circulation by asialoglycoprotein receptor-expressing liver macrophages and hepatocytes.Blood. 2009; 114: 1645-1654Crossref PubMed Scopus (160) Google Scholar Here, we present the first type of human macrothrombocytopenia that is associated with a specific defect of α2,3-sialylation and with strong platelet binding to the ASGP-R. Flow cytometry was performed according to standard protocols.10Marquardt T. Lühn K. Srikrishna G. Freeze H.H. Harms E. Vestweber D. Correction of leukocyte adhesion deficiency type II with oral fucose.Blood. 1999; 94: 3976-3985Crossref PubMed Google Scholar Anti-sLex antibody CSLEX-1 (American Type Culture Collection, Manassas, VA), anti-Lex antibody (BD Pharmingen, Heidelberg, Germany), negative control IgM monoclonal antibody (mAb) (BD Pharmingen), anti-GQ1b/GD3 mAb R24,11Pukel C.S. Lloyd K.O. Travassos L.R. Dippold W.G. Oettgen H.F. Old L.J. GD3, a prominent ganglioside of human melanoma Detection and characterisation by mouse monoclonal antibody.J Exp Med. 1982; 155: 1133-1147Crossref PubMed Scopus (492) Google Scholar anti-PSA mAb 735,12Frosch M. Görgen I. Boulnois G.J. Timmis K.N. Bitter-Suermann D. NZB mouse system for production of monoclonal antibodies to weak bacterial antigens: isolation of an IgG antibody to the polysaccharide capsules of Escherichia coli K1 and group B meningococci.Proc Natl Acad Sci U S A. 1985; 82: 1194-1198Crossref PubMed Scopus (341) Google Scholar anti-Lea antibody (Acris Antibodies, Hiddenhausen, Germany), and anti-GP1b mAb AK2 (Serotec, Düsseldorf, Germany) were used at 10 μg/mL. Biotinylated Aleuria aurantia lectin (AAL), concanavalin A, peanut agglutinin, Sambucus nigra lectin (SNA), and Maackia amurensis lectin II (MAL II) (Vector Laboratories, Burlingame, CA) were used at 2 to 5 μg/mL and detected with phycoerythrin-conjugated streptavidin (Jackson Immunoresearch, West Grove, PA). E- and P-selectin-Fc13Hahne M. Jager U. Isenmann S. Hallmann R. Vestweber D. Five tumor necrosis factor-inducible cell adhesion mechanisms on the surface of mouse endothelioma cells mediate the binding of leukocytes.J Cell Biol. 1993; 121: 655-664Crossref PubMed Scopus (201) Google Scholar were used as described.10Marquardt T. Lühn K. Srikrishna G. Freeze H.H. Harms E. Vestweber D. Correction of leukocyte adhesion deficiency type II with oral fucose.Blood. 1999; 94: 3976-3985Crossref PubMed Google Scholar A total of 0.2 μg murine myelin-associated glycoprotein (MAG)-Fc and human CD22-Fc (prepared as in14Kelm S. Pelz A. Schauer R. Filbin M.T. Tang S. de Bellard M.E. Schnaar R.L. Mahoney J.A. Hartnell A. Bradfield P. Crocker P.R. Sialoadhesin, myelin-associated glycoprotein and CD22 define a new family of sialic acid-dependent adhesion molecules of the immunoglobulin superfamily.Curr Biol. 1994; 4: 965-972Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar), respectively, were incubated with phycoerythrin-conjugated donkey anti-human IgG (0.2 μg; Jackson Immunoresearch) in 50 μL of PBS for 1 hour and added to the cells after 10 minutes of Fc receptor blockage. IL-8 binding to granulocytes was tested by incubating the cells with different concentrations of a fluorescently labeled human IL-8 peptide [K69(CF)]hIL-8(1–77) containing amino acids 1–77 and carboxyfluorescein attached to lysine 6915David R. Machova Z. Beck-Sickinger A.G. Semisynthesis and application of carboxyfluorescein-labelled biologically active human interleukin-8.Biol Chem. 2003; 384: 1619-1630Crossref PubMed Scopus (26) Google Scholar in PBS/0.1% bovine serum albumin for 30 minutes at 4°C before cells were washed, fixed, and then analyzed by flow cytometry. For control, cells were treated with 100 mU/mL neuraminidase (from Vibrio cholerae; Sigma) for 30 minutes at 37°C in PBS/0.1% bovine serum albumin before they were washed three times and were incubated with IL-8. Platelet analysis was done with the following precaution: For comparison of control and patient platelets, we gated on a very small window in the forward scatter/sideward scatter where large normal platelets and small patient platelets overlap in size to exclude size effects. To test for ASGP-R binding, platelets were incubated with 2.5 μg/mL ASGP-R (purified from human liver as described16Treichel U. Schreiter T. Meyer zum Buschenfelde K.H. Stockert R.J. High-yield purification and characterization of human asialoglycoprotein receptor.Protein Expr Purif. 1995; 6: 251-255Crossref PubMed Scopus (18) Google Scholar) for 1 hour at 4°C, washed and incubated with anti-ASGP-R mAb (30201; Calbiochem, San Diego, CA). To distinguish patient and donor platelets within the patient samples, the Lea-positive patient received Lea-negative thrombocytes. Patient and donor platelets were distinguished with an anti-Lea antibody (Acris Antibodies). Values obtained by flow cytometry give mean fluorescence intensity (MFI) ± SD from at least three experiments. Leukocyte rolling was tested with whole human blood in a blood-perfused flow chamber system17Kuwano Y. Spelten O. Zhang H. Ley K. Zarbock A. Rolling on E- or P-selectin induces the extended but not high-affinity conformation of LFA-1 in neutrophils.Blood. 2010; 116: 617-624Crossref PubMed Scopus (136) Google Scholar in which rectangular glass capillaries were coated with 5 μg/mL human E- and 20 μg/mL human P-selectin-Fc proteins (both from R&D Systems, Abingdon, UK), respectively, for 2 hours and then blocked for 1 hour using casein. Slow rolling was obtained by co-immobilizing E- and P-selectin with intercellular cell adhesion molecule-1 (ICAM-1) 3 and 5 μg/mL, respectively). Representative fields of view were recorded for 1 minute using a SW40/0.75 objective and a digital camera (Sensicam QE; Cooke Corporation, Kelheim, Germany). Results were normalized according to the leukocyte numbers of the blood samples. Transferrin was immunoprecipitated from serum and stained.18Niehues R. Hasilik M. Alton G. Korner C. Schiebe-Sukumar M. Koch H.G. Zimmer K.P. Wu R. Harms E. Reiter K. von Figura K. Freeze H.H. Harms H.K. Marquardt T. Carbohydrate-deficient glycoprotein syndrome type Ib Phosphomannose isomerase deficiency and mannose therapy.J Clin Invest. 1998; 101: 1414-1420Crossref PubMed Google Scholar Isoelectric focusing was performed with a Phast electrophoresis system using a gel with a pH range of 5 to 7 (Pharmacia, Freiburg, Germany). Isofocusing of apolipoprotein C-III (ApoCIII) was performed as described19Wopereis S. Grunewald S. Morava E. Penzien J.M. Briones P. Garcia-Silva M.T. Demacker P.N. Huijben K.M. Wevers R.A. Apolipoprotein C-III isofocusing in the diagnosis of genetic defects in O-glycan biosynthesis.Clin Chem. 2003; 49: 1839-1845Crossref PubMed Scopus (113) Google Scholar using a mixture of pharmalytes (4.2 to 4.9 and 2.5 to 5; GE Healthcare, Freiburg, Germany), Western blotting with purified anti-ApoCIII Ab (Rockland, Gilbertsville, PA) and relative quantification of the Enhanced chemiluminescence signal on a Fujifilm Luminescent Image Analyzer (Fuji Photo Film, Tokyo, Japan). Fibroblasts were transfected with mouse α1,3-fucosyltransferase VII DNA in vector pcDNA320Huang M.C. Laskowska A. Vestweber D. Wild M.K. The alpha (1,3)-fucosyltransferase Fuc-TIV, but not Fuc-TVII, generates sialyl Lewis X-like epitopes preferentially on glycolipids.J Biol Chem. 2002; 277: 47786-47795Crossref PubMed Scopus (40) Google Scholar by nucleofection as described.21Helmus Y. Denecke J. Yakubenia S. Robinson P. Luhn K. Watson D.L. McGrogan P.J. Vestweber D. Marquardt T. Wild M.K. Leukocyte adhesion deficiency II patients with a dual defect of the GDP-fucose transporter.Blood. 2006; 107: 3959-3966Crossref PubMed Scopus (63) Google Scholar Total RNA (2 μg) was isolated from blood leukocytes (for mutation analysis) or monocytes (for real-time PCR) using a RNAeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was obtained with SuperScript Reverse Transcriptase III (Invitrogen, Karlsruhe, Germany). Sequencing was done using an ABI Prism 3700 capillary sequencer and BigDye 3.1 (Applied Biosystems, Darmstadt, Germany). Quantitative PCR was performed with appropriate primers in an ABI PRISM 7900HT device (Applied Biosystems) using the QuantiTect SYBR Green PCR Master Mix (Qiagen). Reactions were performed in reference to housekeeping genes cyclophilin A, hALU, and GAPDH and compared to control cDNA samples from four healthy donors. Genomic DNA was prepared from EDTA-treated whole blood samples of the patient and healthy control donors using the QiaAmp Blood Kit (Qiagen). For generation of cDNA, RNA was prepared from whole blood using the PaxGene Blood RNA Kit (Qiagen) or from fibroblasts using the RNAEasy Kit (Qiagen) and was used directly for PCR. All eight exons of the genomic Slc35a1 sequence were amplified with the following forward and reverse primers binding to flanking intron sequences: exon 1, 5′-GCGGGGAGACGCAGTTTACA-3′, 5′-CACTCCCAGCTAGTGGAGGT-3′; exon 2, 5′-GCAATTGGGGCACTCCCTAG-3′, 5′-GCTTTGCAAGCCAGTCAGTC-3′; exon 3 plus 4, 5′-CACTTGAACACGGGAGGTG-3′, 5′-CCTCTTTGGGGACTGTCATC-3′; exon 5, 5′-GGAGTCTTAAGAGGCAGCAC-3′, 5′-GCCGGACTGAGATGGTCAAG-3′; exon 6 plus intron 6 plus exon 7, 5′-GTCATGTGTCACACAACCTAC-3′, 5′-CTAGCACCCCTCGTCTTAAC-3′; exon 8, 5′-CTTGATTTTACCCGCCCTGC-3′, 5′-GTAGACCCCAAACAGGTCTA-3′. PCR conditions were 5 minutes at 94°C, 35 cycles with 1 minute at 94°C, 1 minute at 67°C, 1.5 minutes at 72°C, and 5 minutes at 72°C. PCR of cDNA was performed with primers 5′-GTACAGTGGAAACCAGCCCA-3′ (bp 499 in NCBI accession No. D87969) and 5′-GTAGACCCCAAACAGGTCTA-3′ (bp 1247) amplifying a fragment of 749 bp starting in exon 4 and ending downstream of the stop codon of Slc35a1 (annealing at 68°C for 2 minutes). PCR products were treated with the PCR product presequencing kit (USB Corporation, Cleveland, OH) before they were sequenced as described before.22Kranz C. Jungeblut C. Denecke J. Erlekotte A. Sohlbach C. Debus V. Kehl H.G. Harms E. Reith A. Reichel S. Grobe H. Hammersen G. Schwarzer U. Marquardt T. A defect in dolichol phosphate biosynthesis causes a new inherited disorder with death in early infancy.Am J Hum Genet. 2007; 80: 433-440Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar Isolated platelets were fixed in 2% paraformaldehyde, 0.2% glutaraldehyde in 0.1 mol/L phosphate buffer and were processed further for ultrathin cryosectioning and immunogold labeling as described.23Slot J.W. Geuze H.J. Cryosectioning and immunolabeling.Nat Protoc. 2007; 2: 2480-2491Crossref PubMed Scopus (334) Google Scholar Studies on the patient's cells, bone marrow biopsies, and publication of results were done with written consent of the patient's parents and approval by the Ethics Commission of the University of Münster. We detected the novel disease in a girl who developed spontaneous bleeding within the first day of life. The neonate was diagnosed with thrombocytopenia (12,000 to 17,000 platelets/μL blood; normal range: 140,000 to 300,000/μL) and strong initial neutropenia (120 neutrophils/μL blood; normal range: 1250 to 6500/μL). Whereas the severity of the thrombocytopenia remained stable over more than 8 years since the patient's birth, the neutropenia showed considerable variability, with neutrophil numbers ranging from 70 to 5700/μL and a gradual normalization (see Supplemental Tables S1 and S2 at http://ajp.amjpathol.org). At the age of 8 years, the patient shows neutrophil counts of 1200 to 3300/μL and is thus no longer neutropenic. Higher neutrophil counts were recorded during bacterial infections. Despite variable neutropenia, the child never showed signs of immunodeficiency. Counts of lymphocytes, monocytes, eosinophils, and basophils were always within the normal range (see Supplemental Tables S1 and S2 at http://ajp.amjpathol.org). Starting with day 1 after birth, the thrombocytopenia had to be controlled by weekly platelet transfusions. No further bleeding events were observed under this substitution regime for the 8 years she has been studied. Von Willebrand disease was excluded and no auto- or allo-antibodies against thrombocytes or granulocytes could be detected in the patient. Further details on the phenotype are given in Table 1.Table 1Phenotype of the Novel Thrombocytopenic Disorder Patient: girl, born in 2002 to nonconsanguineous parents, uneventful pregnancy Normal weight, length, and head circumference Petechial bleedings at trunk and legs, bloody vomiting, and small intracerebral bleeding (I°) 12 hours after birth Initial thrombocytopenia of 12,000 thrombocytes per μL No antibodies against GP IIb/IIIa, Ia/IIa, Ib/IX, and HLA in maternal serum No antibodies on the surface of patient platelets, giving no indication of neonatal alloimmune thrombocytopenia Abundant megakaryocytes with normal to decreased size and normal neutrophil precursor cells in bone marrow biopsies Enlarged thrombocytes of heterogenous size with giant thrombocytes nearly as large as erythrocytes in peripheral blood Neutrophil counts below 1000 per μL at 4 weeks of age and below 500 per μL on several occasions thereafter No antibodies against granulocytes detectable Thrombocytopenia and neutropenia unresponsive to corticosteroid treatment for 2 weeks and to immunoglobulin treatment No further bleeding complications due to weekly thrombocyte transfusions Thrombocyte counts constantly below 10,000 per μL upon admission Despite neutropenia no increased frequency of infections No splenomegaly Normal psychomotor development Open table in a new tab Flow cytometry analysis of peripheral blood cells revealed the presence of normal-sized thrombocytes as well as of giant platelets. This was confirmed by electron microscopy (Figure 1). The enlarged platelets had a mean diameter of 5.0 μm with a range of 3.5 to 6.3 μm (control platelets mean: 2.5 μm, range: 1.8 to 2.8 μm) and a calculated mean volume of 65 fL with a range of 30 to 80 fL (control platelets: 8 fL, range: 5 to 12 fL). Ultrastructural analysis showed striking abnormalities of the platelets' open canalicular membrane system (OCS). Here, the multiple segmented channels that form the OCS in normal platelets (Figure 1A) were replaced by one or two large saccular compartments (Figure 1, B and C), strongly indicating a defect in platelet formation. The micrographs also show sphere-like structures within the OCS of the patient platelets, but not of control platelets. Some of these structures appear to be surrounded by membranes. Abnormal membrane blebbing of the OCS may be an explanation for this phenomenon. Bone marrow examination on three separate occasions showed that erythroid and granulocytic maturation were largely normal with a marginal left shift. Interestingly, megakaryocytic hyperplasia with an increased number of micromegakaryocytes was found (not shown). To analyze the molecular cause for this disease, we turned to granulocytes that were obviously also affected but considerably easier to obtain from the patient than platelets. When we analyzed the expression of adhesion molecules in patient granulocytes, we noticed that binding of soluble E- and P-selectin-Fc fusion proteins was reduced by 70% ± 14% and 76% ± 15%, respectively [mean fluorescence intensity (MFI) values ± SD from five independent flow cytometry experiments] (Figure 2A). This defect, however, was not as strong as in leukocyte adhesion deficiency II, where the expression of functional selectin ligands and leukocyte rolling on selectins are virtually absent.10Marquardt T. Lühn K. Srikrishna G. Freeze H.H. Harms E. Vestweber D. Correction of leukocyte adhesion deficiency type II with oral fucose.Blood. 1999; 94: 3976-3985Crossref PubMed Google Scholar, 24Etzioni A. Frydman M. Pollack S. Avidor I. Phillips M.L. Paulson J.C. Gershoni-Baruch R. Brief report: recurrent severe infections caused by a novel leukocyte adhesion deficiency.N Engl J Med. 1992; 327: 1789-1792Crossref PubMed Scopus (444) Google Scholar Indeed, the number of patient and control leukocytes rolling on immobilized E- and P-selectin in flow chamber assays showed no significant difference (Figure 2B). Moreover, the velocities of cells rolling on selectins or on selectins plus intercellular adhesion molecule-1 (slow rolling) were identical for patient and control leukocytes (Figure 2C). These data suggest that the residual selectin ligands that we detected in patient cells are sufficient to preserve selectin-mediated interactions. Finally, we tested binding of interleukin-8 (IL-8) to granulocytes since the interaction of this leukocyte arrest-mediating chemokine was found to be reduced in mice deficient for α2,3-sialyltransferase IV and in human cells treated with neuraminidase.25Frommhold D. Ludwig A. Bixel M.G. Zarbock A. Babushkina I. Weissinger M. Cauwenberghs S. Ellies L.G. Marth J.D. Beck-Sickinger A.G. Sixt M. Lange-Sperandio B. Zernecke A. Brandt E. Weber C. Vestweber D. Ley K. Sperandio M. Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation.J Exp Med. 2008; 205: 1435-1446Crossref PubMed Scopus (60) Google Scholar Figure 2D shows that binding of human IL-8 to patient granulocytes over a concentration range of 10 to 90 nmol/L was only marginally reduced (MFI: 84% ± 29% of control values, n = 9). In fact, only at the highest IL-8 concentration a reduction was detected (an example is shown in the Figure 2D). Taken together, the data on selectin-mediated rolling and IL-8 binding are consistent with the finding that the patient shows normal immunity. Selectin ligands carry fucosylated and sialylated glycostructures that are identical with or similar to sialyl-Lewis X (sLex) and are required for selectin binding. We found that patient granulocytes exhibited strongly reduced surface expression of sLex (19% ± 9% of control MFI, n = 8) and increased expression of its nonsialylated form Lex (463% ± 96% of control MFI, n = 8) (Figure 3A). Similar results were obtained for blood monocytes (not shown). We then studied lectin binding to granulocytes and found normal binding of concanavalin A and AAL, which are specific for α-linked mannose and fucose, respectively (not shown). However, the cells showed increased binding of peanut agglutinin (PNA) which binds to the nonsialylated, but not to the sialylated, form of the T antigen (galactosyl-β1,3-N-acetylgalactosamine), suggesting a defect in T antigen sialylation (see Supplemental Figure S1 at http://ajp.amjpathol.org). More interestingly, we found that binding of Maackia amurensis lectin II (MAL II), which preferentially recognizes sialic acid linked to galactose in α2,3-linkage, was reduced (Figure 3B). In contrast, binding of SNA, which preferentially recognizes α2,6-linked sialic acid, was normal (Figure 3B). Together with the reduced expression of the α2,3-sialylated sLex, these data strongly suggested a partial α2,3-sialylation defect. Hypo-α2,3-sialylation in granulocytes was confirmed by reduced binding of recombinant myelin-associated glycoprotein (MAG/Siglec-4), which is specific for α2,3-linked sialic acid, whereas binding of α2,6-sialylation-specific CD22 (Siglec-2) was normal (Figure 3C). This pattern of defective α2,3-sialylation and normal α2,6-sialylation was also found in peripheral blood monocytes (not shown). In control lymphocytes, Lex and sLex were hardly detectable, but MAL II and MAG readily bound and gave equal signals in control and patient cells, showing that the defect in α2,3-sialylation is not present in these cells (data not shown). As skin fibroblasts are devoid of sLex, we transfected them with a plasmid containing the cDNA sequence coding for α1,3-fucosyltransferase VII. This led to virtually equal expression of sLex in healthy and patient fibroblasts (not shown), demonstrating that skin fibroblasts are not affected by the disease and that the defect is cell-specific. In contrast to α2,3-sialylation, the third type of sialic acid linkage, α2,8-sialylation, was found to be normal in granulocytes as judged by normal binding of mAb CGM3, which reacts with the α2,8-sialylated gangliosides GQ1b and GD3 (Figure 3D). Binding of mAb 735, which is specific for α2,8-sialylated polysialic acid, bound very weakly to control granulocytes (not shown). However, it showed detectable and equal binding to control and patient monocytes (Figure 3D), further excluding a defect in α2,8-sialylation. Flow cytometry analysis of control and patient thrombocytes showed that E- and P-selectin as well as antibodies against sLex, Lex, GQ1b/GD3, and polysialic acid did not bind to these cells. However, we found that, as in granulocytes, binding of α2,3-sialylation–specific MAL II and MAG, but not of α2,6-sialylation–specific SNA and CD22, to patient platelets was reduced (Figure 4, A and B). Binding of fucose-specific AAL was normal (Figure 4C). Changes in the sialylation patterns of serum transferrin and/or apolipoprotein C-III had been shown for all types of CDG-I as well as CDG-IIa, -d, -e, -f.26Wopereis S. Grunewald S. Huijben K.M. Morava E. Mollicone R. van Engelen B.G. Lefeber D.J. Wevers R.A. Transferrin and apolipoprotein C-III isofocusing are complementary in the diagnosis of N- and O-glycan biosynthesis defects.Clin Chem. 2007; 53: 180-187Crossref PubMed Scopus (53) Google Scholar However, isoelectric focusing of the patient's serum transferrin and apolipoprotein C-III revealed normal sialylation (Figure 5 and Table 2). In addition, defects in the generation of the lipid-linked oligosaccharide (LLO), which are indicative of CDG-I, were excluded by high-performance liquid chromatography analysis (see Supplemental Figure S2 at http://ajp.amjpathol.org). Taken together, these data show that the novel disease is characterized, not by a general sialylation deficiency, but rather by a specific defect in α2,3-sialylation in a restricted set of cells.Table 2Isoelectric Focusing of Apolipoprotein CIIIPercentage of ApoCIII isoforms⁎Three independently collected serum samples of the patient and samples of the patient's mother were subjected to isoelectrical focusing. Results obtained for a CDG-IIf patient26 are shown for comparison. The samples of the patient and her mother show isoform distributions that are within the respective reference ranges, whereas the CDG-IIf results indicate hyposialylation of ApoCIII as seen from the shift of disialylated ApoCIII-2 to monosialylated ApoCIII-1.ApoCIII-0 (%)ApoCIII-1 (%)ApoCIII-2 (%)Patient, sample 15.368.426.4Patient, sample 26.560.433.2Patient, sample 39.756.533.7CDG-IIf patient6.078.016.0Reference range (age: 1–18 yr)0–11.633.1–66.927.4–60.0Patient's mother8.159.532.4Reference range (age: >18 yr)2.6–18.942.9–69.223.2–50.0 Three independently collected serum samples of the patient and samples of the patient's mother were subjected to isoelectrical focusing. Results obtained for a CDG-IIf patient26Wopereis S. Grunewald S. Huijben K.M. Morava E. Mollicone R. van Engelen B.G. Lefeber D.J. Wevers R.A. Transferrin and apolipoprotein C-III isofocusing are complementary in the diagnosis of N- and O-glycan biosynthesis defects.Clin Chem. 2007; 53: 180-187Crossref PubMed Scopus (53) Google Scholar are shown for comparison. The samples of the patient and her mother show isoform distributions that are within the respective reference ranges, whereas the CDG-IIf results indicate hyposialylation of ApoCIII as seen from the shift of disialylated ApoCIII-2 to monosialylated ApoCIII-1. Open table in a new tab We performed a l
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