Resistance of B16 Melanoma Cells to CD47-induced Negative Regulation of Motility as a Result of Aberrant N-Glycosylation of SHPS-1

Ogura, Takeshi; Noguchi, Tetsuya; Murai-Takebe, Reiko; Hosooka, Tetsuya; Honma, Nakayuki; Kasuga, Masato

doi:10.1074/jbc.m310276200

Cited by 25 publications

(26 citation statements)

References 52 publications

(58 reference statements)

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“…Removal of sugars on human CD47 is found to lead to enhanced interactions with soluble SIRP␣ just as deglycosylated soluble SIRP␣ interacts more strongly with CD47 (Fig. 2), consistent with past reports (35).…”

Section: Discussionsupporting

confidence: 91%

“…These results are consistent with the notion that hyperglycosylation of either protein could suppress detectable association, as seen with B16 melanoma cells that express hyperglycosylated SIRP␣ (35). However, the role of sugar type cannot be ruled out, since B16 melanoma cells also overexpress ␤-1,6-N-acetylglucosaminyltransferase (GnT-V/Mgat5) that introduces ␤-1,6-GlcNAc-branched N-glycans strongly linked with increased cell invasiveness and metastatic potential (49 -51).…”

Section: Discussionsupporting

confidence: 90%

“…SIRP␣ glycosylation is already known to moderate its interactions with CD47 (25,35). CD47 glycosylation has unknown roles in function or even export in mammalian cells.…”

Section: Display Of Wild-type Cd47 and Cd47-gfp Fusion-full-mentioning

confidence: 99%

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Phylogenetic Divergence of CD47 Interactions with Human Signal Regulatory Protein α Reveals Locus of Species Specificity

Subramanian

Boder

Discher

2007

Journal of Biological Chemistry

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Cell-cell interactions between ubiquitously expressed integrinassociated protein (CD47) and its counterreceptor signal regulatory protein (SIRP␣) on phagocytes regulate a wide range of adhesive signaling processes, including the inhibition of phagocytosis as documented in mice. We show that CD47-SIRP␣ binding interactions are different between mice and humans, and we exploit phylogenetic divergence to identify the species-specific binding locus on the immunoglobulin domain of human CD47. All of the studies are conducted in the physiological context of membrane protein display on Chinese hamster ovary (CHO) cells. Novel quantitative flow cytometry analyses with CD47-green fluorescent protein and soluble human SIRP␣ as a probe show that neither human CD47 nor SIRP␣ requires glycosylation for interaction. Human CD47-expressing CHO cells spread rapidly on SIRP␣-coated glass surfaces, correlating well with the spreading of primary human T cells. In contrast, CHO cells expressing mouse CD47 spread minimally and show equally weak binding to soluble human SIRP␣. Further phylogenetic analyses and multisite substitutions of the CD47 Ig domain show that human to cow mutation of a cluster of seven residues on adjacent strands near the middle of the domain decreases the association constant for human SIRP␣ to about onethird that of human CD47. Direct tests of cell-cell adhesion between human monocytes and CD47-displaying CHO cells affirm the species specificity as well as the importance of the newly identified binding locus in cell-cell interactions.Cell-cell interactions are of course critical in immune function but can have important and revealing differences between species as well as nonlinear dependences on molecular parameters, such as affinity. We explore such general issues with integrin-associated protein (IAP), 2 or CD47, which is a ubiquitous but unique immunoglobulin superfamily receptor with a single Ig domain, a pentaspan transmembrane segment, and a variably spliced cytoplasmic tail (1). The known functional roles of CD47, originally limited to integrin activation (2), have expanded considerably since the identification of SIRP␣ as a physiological ligand (3-6). SIRP␣ is another Ig superfamily member with three Ig domains and a single span transmembrane segment. SIRP␣ is also broadly expressed, present at high levels on myeloid lineages (monocytes, neutrophils) and neurons but absent from many if not most other cells (7). Both CD47 and SIRP␣ are found in many mammals, but the differences in sequence (Fig. 1A) across species have yet to be evaluated. CD47-SIRP␣ interactions regulate transmigration of monocytes and neutrophils (8 -11) with severe impairment in neutrophil recruitment to sites of infection in CD47 knock-out mice (12). CD47 on "self " red cells, platelets, lymphocytes, and other nonapoptotic cells inhibits clearance by macrophages in mice by signaling through SIRP␣ (13-16). However, in humans as opposed to mice, severe reductions of CD47 levels on red cells, as found on various Rh-deficient patient ...

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Section: Discussionsupporting

confidence: 91%

Section: Discussionsupporting

confidence: 90%

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Phylogenetic Divergence of CD47 Interactions with Human Signal Regulatory Protein α Reveals Locus of Species Specificity

Subramanian

Boder

Discher

2007

Journal of Biological Chemistry

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“…Fourth, this ubiquitin ligase degrades the adhesion receptor SHPS-1, which may be involved in tumor metastasis in melanomas and other cancers. [56][57][58] Finally, we show here that FBG1 interacts with APC2 and Cul7, both of which play important roles in lower levels. Second, in contrast to other FBG family members, FBG1 is not expressed during embryonic development and its expression increases in the brain during the first ten months of life.…”

Section: Scf Ubiquitin Ligase Complex (Pdb Code 1ldk) Is Shown For Rementioning

confidence: 95%

FBG1 is a promiscuous ubiquitin ligase that sequesters APC2 and causes S-phase arrest

Wen¹,

Kim

Fuentes

et al. 2010

Cell Cycle

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“…Glycosylation has been implicated as a determinant of binding specificity in at least one binding partner of CD47 (van den Nieuwenhof et al, 2001;Ogura et al, 2004), but analogous information is lacking for CD47 itself. Using yeast surface display as a platform, we describe here the roles that variant glycosylation and disulfide bond formation play in determining surface expression levels and conformation of CD47's Ig domain (IgCD47).…”

Section: Cd47 or Integrin Associated Protein (Iap) Is A Prototypical mentioning

confidence: 99%

Post‐translational regulation of expression and conformation of an immunoglobulin domain in yeast surface display

Parthasarathy

Subramanian

Boder

et al. 2005

Biotech & Bioengineering

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Display of heterologous proteins on the surface of Saccharomyces cerevisiae is increasingly being exploited for directed evolution because of straightforward cell screens. However, yeast post-translationally modifies proteins in ways that must be factored into library engineering and refinement. Here, we express the extracellular immunoglobulin domain of an ubiquitous mammalian membrane protein, CD47, which is implicated in cancer, immunocompatibility, and motility. CD47 has multiple sites of glycosylation and a core disulfide bond. We assess the effects of both of these post-translational modifications on expression and antibody binding. CD47's extracellular domain is fused to the yeast mating protein Aga2p on the cell wall, and the resulting fusion protein binds several key antibodies, including a conformation-sensitive antibody. Site-bysite mutagenesis of CD47's five N-linked glycosylation sites progressively decreases expression levels on yeast, but folding appears stable. Cysteine mutations disrupt the expected core disulfide, and also decrease protein expression levels, though not to the extent seen with complete deglycosylation. However, with the core disulfide mutants, antibody binding proves to be lower than expression levels might indicate and glycosylation is clearly reduced compared to wild-type. The results indicate that glycosylation regulates heterologous display on yeast more than core disulfides do and thus suggest bounds on directed evolution by post-translational processing. ABSTRACTDisplay of heterologous proteins on the surface of Saccharomyces cerevisiae is increasingly being exploited for directed evolution because of straightforward cell screens. However, yeast post-translationally modifies proteins in ways that must be factored into library engineering and refinement. Here, we express the extracellular immunoglobulin domain of an ubiquitous mammalian membrane protein, CD47, which is implicated in cancer, immunocompatibility, and motility. CD47 has multiple sites of glycosylation and a core disulfide bond. We assess the effects of both of these post-translational modifications on expression and antibody binding.

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Resistance of B16 Melanoma Cells to CD47-induced Negative Regulation of Motility as a Result of Aberrant N-Glycosylation of SHPS-1

Abstract: The adhesion receptor SHPS-1 activates the proteintyrosine-phosphatase SHP-2 and thereby promotes integrin-mediated reorganization of the cytoskeleton.

Cited by 25 publications

References 52 publications

Phylogenetic Divergence of CD47 Interactions with Human Signal Regulatory Protein α Reveals Locus of Species Specificity

Phylogenetic Divergence of CD47 Interactions with Human Signal Regulatory Protein α Reveals Locus of Species Specificity

FBG1 is a promiscuous ubiquitin ligase that sequesters APC2 and causes S-phase arrest

Post‐translational regulation of expression and conformation of an immunoglobulin domain in yeast surface display

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