N-Glycosylation of the Human Granulocyte-Macrophage Colony-stimulating Factor Receptor α Subunit Is Essential for Ligand Binding and Signal Transduction

Ding, Dawn Xiao-Hong; Vera, Juan Carlos; Heaney, Mark L.; Golde, David W.

doi:10.1074/jbc.270.41.24580

Cited by 57 publications

(32 citation statements)

References 45 publications

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“…Human GM-CSF exhibits variable N-linked glycosylation site occupancy, which plays an important role in its biological activity. Indeed, it has been demonstrated that there is an inverse correlation between biological activity and the extent of N-glycosylation, suggesting that N-linked glycans down-regulate the bioactivity of the molecule (36,37). The 85-kDa isoform of CD44 on hemopoietic cells, on the other hand, acts as a ligand for hyaluronan produced by endothelial cells when it is sulfated on O-linked oligosaccharides in response to TNF-␣ stimulation (38).…”

Section: Discussionmentioning

confidence: 99%

CD164 Monoclonal Antibodies That Block Hemopoietic Progenitor Cell Adhesion and Proliferation Interact with the First Mucin Domain of the CD164 Receptor

Doyonnas

Chan

Butler

et al. 2000

The Journal of Immunology

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The novel sialomucin, CD164, functions as both an adhesion receptor on human CD34+ cell subsets in bone marrow and as a potent negative regulator of CD34+ hemopoietic progenitor cell proliferation. These diverse effects are mediated by at least two functional epitopes defined by the mAbs, 103B2/9E10 and 105A5. We report here the precise epitope mapping of these mAbs together with that of two other CD164 mAbs, N6B6 and 67D2. Using newly defined CD164 splice variants and a set of soluble recombinant chimeric proteins encoded by exons 1–6 of the CD164 gene, we demonstrate that the 105A5 and 103B2/9E10 functional epitopes map to distinct glycosylated regions within the first mucin domain of CD164. The N6B6 and 67D2 mAbs, in contrast, recognize closely associated and complex epitopes that rely on the conformational integrity of the CD164 molecule and encompass the cysteine-rich regions encoded by exons 2 and 3. On the basis of their sensitivities to reducing agents and to sialidase, O-sialoglycoprotease, and N-glycanase treatments, we have characterized CD164 epitopes and grouped them into three classes by analogy with CD34 epitope classification. The class I 105A5 epitope is sialidase, O-glycosidase, and O-sialoglycoprotease sensitive; the class II 103B2/9E10 epitope is N-glycanase, O-glycosidase, and O-sialoglycoprotease sensitive; and the class III N6B6 and 67D2 epitopes are not removed by such enzyme treatments. Collectively, this study indicates that the previously observed differential expression of CD164 epitopes in adult tissues is linked with cell type specific post-translational modifications and suggests a role for epitope-associated carbohydrate structures in CD164 function.

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Section: Discussionmentioning

confidence: 99%

CD164 Monoclonal Antibodies That Block Hemopoietic Progenitor Cell Adhesion and Proliferation Interact with the First Mucin Domain of the CD164 Receptor

Doyonnas

Chan

Butler

et al. 2000

The Journal of Immunology

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“…The other consists of biological functions. Recent reports have provided evidence that N-linked glycosylation may be required for ligand recognition or signaling (27)(28)(29)(30). However, what role N-linked glycosylation plays in ligand binding and signaling remains uncertain.…”

Section: Discussionmentioning

confidence: 99%

N-Linked Glycosylations at Asn26 and Asn114 of Human MD-2 Are Required for Toll-Like Receptor 4-Mediated Activation of NF-κB by Lipopolysaccharide

Ohnishi

Muroi

Tanamoto

2001

The Journal of Immunology

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MD-2 is physically associated with Toll-like receptor 4 (TLR4) and is required for TLR4-mediated LPS signaling. Western blotting analysis revealed the presence of three forms of human (h)MD-2 with different electrophoretic mobilities. After N-glycosidase treatment of the cellular extract prepared from cells expressing hMD-2, only a single form with the fastest mobility was detected. Mutation of either one of two potential glycosylation sites (Asn26 and Asn114) of MD-2 resulted in the disappearance of the slowest mobility form, and only the fastest form was detected in hMD-2 carrying mutations at both Asn26 and Asn114. Although these mutants were expressed on the cell surface and maintained its ability to associate with human TLR4, these mutations or tunicamycin treatment substantially impaired the ability of MD-2 to complement TLR4-mediated activation of NF-κB by LPS. LPS binding to cells expressing CD14, TLR4, and MD-2 was unaffected by these mutations. These observations demonstrate that hMD-2 undergoes N-linked glycosylation at Asn26 and Asn114, and that these glycosylations are crucial for TLR4-mediated signal transduction of LPS.

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“…Western analysis of parallel samples for actin ensured equal loading of cell lysates. The band corresponding to GM-CSF receptor a glycoprotein is broad because of glycosylation at multiple sites (43). Note the absence of GM-CSF receptor a in patient B but not her parents or control.…”

Section: (Not Shown) (B)mentioning

confidence: 99%

Hereditary Pulmonary Alveolar Proteinosis

Suzuki¹,

Sakagami²,

Young³

et al. 2010

Am J Respir Crit Care Med

153

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Rationale: We identified a 6-year-old girl with pulmonary alveolar proteinosis (PAP), impaired granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor function, and increased GM-CSF. Objectives: Increased serum GM-CSF may be useful to identify individuals with PAP caused by GM-CSF receptor dysfunction. Methods: We screened 187 patients referred to us for measurement of GM-CSF autoantibodies to diagnose autoimmune PAP. Five were children with PAP and increased serum GM-CSF but without GM-CSF autoantibodies or any disease causing secondary PAP; all were studied with family members, subsequently identified patients, and controls. Measurement and Main Results: Eight children (seven female, one male) were identified with PAP caused by recessive CSF2RA mutations. Six presented with progressive dyspnea of insidious onset at 4.8 6 1.6 years and two were asymptomatic at ages 5 and 8 years. Radiologic and histopathologic manifestations were similar to those of autoimmune PAP. Molecular analysis demonstrated that GM-CSF signaling was absent in six and severely reduced in two patients. The GM-CSF receptor b chain was detected in all patients, whereas the a chain was absent in six and abnormal in two, paralleling the GM-CSF signaling defects. Genetic analysis revealed multiple distinct CSF2RA abnormalities, including missense, duplication, frameshift, and nonsense mutations; exon and gene deletion; and cryptic alternative splicing. All symptomatic patients responded well to whole-lung lavage therapy. Conclusions: CSF2RA mutations cause a genetic form of PAP presenting as insidious, progressive dyspnea in children that can be diagnosed by a combination of characteristic radiologic findings and blood tests and treated successfully by whole-lung lavage.

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N-Glycosylation of the Human Granulocyte-Macrophage Colony-stimulating Factor Receptor α Subunit Is Essential for Ligand Binding and Signal Transduction

Cited by 57 publications

References 45 publications

CD164 Monoclonal Antibodies That Block Hemopoietic Progenitor Cell Adhesion and Proliferation Interact with the First Mucin Domain of the CD164 Receptor

CD164 Monoclonal Antibodies That Block Hemopoietic Progenitor Cell Adhesion and Proliferation Interact with the First Mucin Domain of the CD164 Receptor

N-Linked Glycosylations at Asn26 and Asn114 of Human MD-2 Are Required for Toll-Like Receptor 4-Mediated Activation of NF-κB by Lipopolysaccharide

Hereditary Pulmonary Alveolar Proteinosis

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