scholarly journals Regulation of CD44 binding to hyaluronan by glycosylation of variably spliced exons.

1995 ◽  
Vol 131 (6) ◽  
pp. 1623-1633 ◽  
Author(s):  
K L Bennett ◽  
B Modrell ◽  
B Greenfield ◽  
A Bartolazzi ◽  
I Stamenkovic ◽  
...  
Keyword(s):  
Regulatory Mechanism ◽  
Protein Glycosylation ◽  
Differential Splicing ◽  
Binding Studies ◽  
Lectin Activity ◽  
Cd44 Isoforms ◽  
Binding Function ◽  
Glycosylation Inhibitor ◽  
Immunoglobulin Domain ◽  
Leukocyte Homing

The hyaluronan (HA)-binding function (lectin function) of the leukocyte homing receptor, CD44, is tightly regulated. Herein we address possible mechanisms that regulate CD44 isoform-specific HA binding. Binding studies with melanoma transfectants expressing CD44H, CD44E, or with soluble immunoglobulin fusions of CD44H and CD44E (CD44H-Rg, CD44E-Rg) showed that although both CD44 isoforms can bind HA, CD44H binds HA more efficiently than CD44E. Using CD44-Rg fusion proteins we show that the variably spliced exons in CD44E, V8-V10, specifically reduce the lectin function of CD44, while replacement of V8-V10 by an ICAM-1 immunoglobulin domain restores binding to a level comparable to that of CD44H. Conversely, CD44 bound HA very weakly when exons V8-V10 were replaced with a CD34 mucin domain, which is heavily modified by O-linked glycans. Production of CD44E-Rg or incubation of CD44E-expressing transfectants in the presence of an O-linked glycosylation inhibitor restored HA binding to CD44H-Rg and to cell surface CD44H levels, respectively. We conclude that differential splicing provides a regulatory mechanism for CD44 lectin function and that this effect is due in part to O-linked carbohydrate moieties which are added to the Ser/Thr rich regions encoded by the variably spliced CD44 exons. Alternative splicing resulting in changes in protein glycosylation provide a novel mechanism for the regulation of lectin activity.

scholarly journals CD44 isoforms containing exon V3 are responsible for the presentation of heparin-binding growth factor.

1995 ◽  
Vol 128 (4) ◽  
pp. 687-698 ◽  
Author(s):  
K L Bennett ◽  
D G Jackson ◽  
J C Simon ◽  
E Tanczos ◽  
R Peach ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factors ◽  
Growth Factor ◽  
Binding Proteins ◽  
Vascular Endothelial Cells ◽  
Heparin Binding ◽  
Binding Studies ◽  
Cd44 Isoforms ◽  
Cultured Endothelial Cells ◽  
Alternatively Spliced

Glycosaminoglycan-modified isoforms of CD44 have been implicated in growth factor presentation at sites of inflammation. In the present study we show that COS cell transfectants expressing CD44 isoforms containing the alternatively spliced exon V3 are modified with heparan sulfate (HS). Binding studies with three HS-binding growth factors, basic-fibroblast growth factor (b-FGF), heparin binding-epidermal growth factor (HB-EGF), and amphiregulin, showed that the HS-modified CD44 isoforms are able to bind to b-FGF and HB-EGF, but not AR. b-FGF and HB-EGF binding to HS-modified CD44 was eliminated by pretreating the protein with heparitinase or by blocking with free heparin. HS-modified CD44 immunoprecipitated from keratinocytes, which express a CD44 isoform containing V3, also bound to b-FGF. We examined whether HS-modified CD44 isoforms were expressed by activated endothelial cells where they might present HS-binding growth factors to leukocytes during an inflammatory response. PCR and antibody-binding studies showed that activated cultured endothelial cells only express the CD44H isoform which does not contain any of the variably spliced exons including V3. Immunohistological studies with antibodies directed to CD44 extracellular domains encoded by the variably spliced exons showed that vascular endothelial cells in inflamed skin tissue sections do not express CD44 spliced variants. Keratinocytes, monocytes, and dendritic cells in the same specimens were found to express variably spliced CD44. 35SO4(-2)-labeling experiments demonstrated that activated cultured endothelial cells do not express detectable levels of chondroitin sulfate or HS-modified CD44. Our results suggest that one of the functions of CD44 isoforms expressing V3 is to bind and present a subset of HS-binding proteins. Furthermore, it is probable that HS-modified CD44 is involved in the presentation of HS-binding proteins by keratinocytes in inflamed skin. However, our data suggests that CD44 is not likely to be the proteoglycan principally involved in presenting HS-binding growth factors to leukocytes on the vascular cell wall.

scholarly journals Identification of the Neuroblastoma-amplified Gene Product as a Component of the Syntaxin 18 Complex Implicated in Golgi-to-Endoplasmic Reticulum Retrograde Transport

2009 ◽  
Vol 20 (11) ◽  
pp. 2639-2649 ◽  
Author(s):  
Takehiro Aoki ◽  
Sarah Ichimura ◽  
Ayano Itoh ◽  
Mami Kuramoto ◽  
Takashi Shinkawa ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Retrograde Transport ◽  
Protein Glycosylation ◽  
Snare Complex ◽  
Binding Studies ◽  
Protein Receptor ◽  
Peripheral Membrane Protein ◽  
A Subunit ◽  
Membrane Components ◽  
Weak Similarity

Syntaxin 18, a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) protein implicated in endoplasmic reticulum (ER) membrane fusion, forms a complex with other SNAREs (BNIP1, p31, and Sec22b) and several peripheral membrane components (Sly1, ZW10, and RINT-1). In the present study, we showed that a peripheral membrane protein encoded by the neuroblastoma-amplified gene (NAG) is a subunit of the syntaxin 18 complex. NAG encodes a protein of 2371 amino acids, which exhibits weak similarity to yeast Dsl3p/Sec39p, an 82-kDa component of the complex containing the yeast syntaxin 18 orthologue Ufe1p. Under conditions favoring SNARE complex disassembly, NAG was released from syntaxin 18 but remained in a p31-ZW10-RINT-1 subcomplex. Binding studies showed that the extreme N-terminal region of p31 is responsible for the interaction with NAG and that the N- and the C-terminal regions of NAG interact with p31 and ZW10-RINT-1, respectively. Knockdown of NAG resulted in a reduction in the expression of p31, confirming their intimate relationship. NAG depletion did not substantially affect Golgi morphology and protein export from the ER, but it caused redistribution of Golgi recycling proteins accompanied by a defect in protein glycosylation. These results together suggest that NAG links between p31 and ZW10-RINT-1 and is involved in Golgi-to-ER transport.

scholarly journals Simultaneous analyses of N-linked and O-linked glycans of ovarian cancer cells using solid-phase chemoenzymatic method

2017 ◽  
Vol 14 (1) ◽  
Author(s):  
Shuang Yang ◽  
Naseruddin Höti ◽  
Weiming Yang ◽  
Yang Liu ◽  
Lijun Chen ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Solid Phase ◽  
Biological Activities ◽  
Protein Glycosylation ◽  
Ovarian Cancer Cells ◽  
Biological Studies ◽  
Glycosylation Inhibitor ◽  
Isobaric Tags ◽  
Glycosylation Inhibition

Abstract Background Glycans play critical roles in a number of biological activities. Two common types of glycans, N-linked and O-linked, have been extensively analyzed in the last decades. N-glycans are typically released from glycoproteins by enzymes, while O-glycans are released from glycoproteins by chemical methods. It is important to identify and quantify both N- and O-linked glycans of glycoproteins to determine the changes of glycans. Methods The effort has been dedicated to study glycans from ovarian cancer cells treated with O-linked glycosylation inhibitor qualitatively and quantitatively. We used a solid-phase chemoenzymatic approach to systematically identify and quantify N-glycans and O-glycans in the ovarian cancer cells. It consists of three steps: (1) immobilization of proteins from cells and derivatization of glycans to protect sialic acids; (2) release of N-glycans by PNGase F and quantification of N-glycans by isobaric tags; (3) release and quantification of O-glycans by β-elimination in the presence of 1-phenyl-3-methyl-5-pyrazolone (PMP). Results We used ovarian cancer cell lines to study effect of O-linked glycosylation inhibitor on protein glycosylation. Results suggested that the inhibition of O-linked glycosylation reduced the levels of O-glycans. Interestingly, it appeared to increase N-glycan level in a lower dose of the O-linked glycosylation inhibitor. The sequential release and analyses of N-linked and O-linked glycans using chemoenzymatic approach are a platform for studying N-glycans and O-glycans in complex biological samples. Conclusion The solid-phase chemoenzymatic method was used to analyze both N-linked and O-linked glycans sequentially released from the ovarian cancer cells. The biological studies on O-linked glycosylation inhibition indicate the effects of O-glycosylation inhibition to glycan changes in both O-linked and N-linked glycan expression.

Missense mutations in the β3 subunit have a different impact on the expression and function between αIIbβ3 and αvβ3

Blood ◽  
2002 ◽  
Vol 99 (3) ◽  
pp. 931-938 ◽  
Author(s):  
Seiji Tadokoro ◽  
Yoshiaki Tomiyama ◽  
Shigenori Honda ◽  
Hirokazu Kashiwagi ◽  
Satoru Kosugi ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Regulatory Mechanism ◽  
Japanese Patients ◽  
Enzyme Linked Immunosorbent Assay ◽  
Missense Mutations ◽  
Glanzmann Thrombasthenia ◽  
Binding Function ◽  
Expression Studies ◽  
293 Cells ◽  
And Function

Abstract αIIbβ3 and αvβ3 belong to the β3integrin subfamily. Although the β3 subunit is a key regulator for the biosynthesis of β3 integrins, it remains obscure whether missense mutations in β3 may induce the same defects in both αIIbβ3 and αvβ3. In this study, it is revealed that thrombasthenic platelets with a His280Pro mutation in β3, which is prevalent in Japanese patients with Glanzmann thrombasthenia, did contain significant amounts of αvβ3 (about 50% of control) using sensitive enzyme-linked immunosorbent assay. Expression studies showed that the His280Proβ3 mutation impaired αIIbβ3 expression but not αvβ3 expression in 293 cells. To extend these findings, the effects of several β3 missense mutations leading to an impaired αIIbβ3expression on αvβ3 function as well as expression was examined: Leu117Trp, Ser162Leu, Arg216Gln, Cys374Tyr, and a newly created Arg216Gln/Leu292Ser mutation. Leu117Trp and Cys374Tyr β3 mutations did impair αvβ3 expression, while Ser162Leu, Arg216Gln, and Arg216Gln/Leu292Ser mutations did not. With regard to ligand binding function, Ser162Leu mutation induced especially distinct effects between 2 β3 integrins: it markedly impaired ligand binding to αIIbβ3 but not to αvβ3 at all. These data clearly demonstrate that the biosynthesis and the ligand binding function of αIIbβ3 and those of αvβ3 are regulated in part by different mechanisms. Present data would be a clue to elucidate the regulatory mechanism of expression and function of β3 integrins.

scholarly journals Impact of Truncated O-glycans in Gastric-Cancer-Associated CD44v9 Detection

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 264 ◽  
Author(s):  
Inês B. Moreira ◽  
Filipe Pinto ◽  
Catarina Gomes ◽  
Diana Campos ◽  
Celso A. Reis
Keyword(s):  
Gastric Cancer ◽  
Monoclonal Antibodies ◽  
Protein Detection ◽  
Protein Glycosylation ◽  
Major Protein ◽  
Cd44 Isoforms ◽  
Cd44 Variant ◽  
Variant Isoforms ◽  
Cancer Models ◽  
Protein Splice

CD44 variant isoforms are often upregulated in cancer and associated with increased aggressive tumor phenotypes. The CD44v9 is one of the major protein splice variant isoforms expressed in human gastrointestinal cancer cells. Immunodetection of CD44 isoforms like CD44v9 in tumor tissue is almost exclusively performed by using specific monoclonal antibodies. However, the structural variability conferred by both the alternative splicing and CD44 protein glycosylation is disregarded. In the present work, we have evaluated the role of O-glycosylation using glycoengineered gastric cancer models in the detection of CD44v9 by monoclonal antibodies. We demonstrated, using different technical approaches, that the presence of immature O-glycan structures, such as Tn and STn, enhance CD44v9 protein detection. These findings can have significant implications in clinical applications mainly at the detection and targeting of this cancer-related CD44v9 isoform and highlight the utmost importance of considering glycan structures in cancer biomarker detection and in therapy targeting.

scholarly journals Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

2021 ◽  
Author(s):  
David S. Roberts ◽  
Morgan W. Mann ◽  
Jake A. Melby ◽  
Eli J. Larson ◽  
Yanlong Zhu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Domain ◽  
Protein Glycosylation ◽  
Ion Cyclotron Resonance ◽  
Top Down ◽  
S Protein ◽  
Viral Binding ◽  
Binding Function ◽  
Protein Receptor ◽  
Top Down Mass Spectrometry

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry and the S protein glycosylation is strongly implicated in altering viral binding/function and infectivity. However, the structures and relative abundance of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis. Here, we report the complete structural characterization of intact O-glycan proteoforms using native top-down mass spectrometry (MS). By combining trapped ion mobility spectrometry (TIMS), which can separate the protein conformers of S-RBD and analyze their gas phase structural variants, with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS analysis, the O-glycoforms of the S-RBD are comprehensively characterized, so that seven O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that native top-down MS can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants, as well as other O-glycoproteins in general.

scholarly journals Receptor protein tyrosine phosphatase PTPmu associates with cadherins and catenins in vivo.

1995 ◽  
Vol 130 (4) ◽  
pp. 977-986 ◽  
Author(s):  
S M Brady-Kalnay ◽  
D L Rimm ◽  
N K Tonks
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Receptor Protein ◽  
Immunocytochemical Staining ◽  
Beta Catenin ◽  
Binding Studies ◽  
Intracellular Domain ◽  
Protein Tyrosine ◽  
Immunoglobulin Domain

The extracellular segment of the receptor-type type protein tyrosine phosphatase PTPmu, possesses an MAM domain, an immunoglobulin domain, and four fibronectin type-III repeats. It binds homophilically, i.e., PTPmu on the surface of one cell binds to PTPmu on an apposing cell, and the binding site lies within the immunoglobulin domain. The intracellular segment of PTPmu has two PTP domains and a juxtamembrane segment that is homologous to the conserved intracellular domain of the cadherins. In cadherins, this segment interacts with proteins termed catenins to mediate association with the actin cytoskeleton. In this article, we demonstrate that PTPmu associates with a complex containing cadherins, alpha- and beta-catenin in mink lung (MvLu) cells, and in rat heart, lung, and brain tissues. Greater than 80% of the cadherin in the cell is cleared from Triton X-100 lysates of MvLu cells after immunoprecipitation with antibodies to PTPmu; however, the complex is dissociated when lysates are prepared in more stringent, SDS-containing RIPA buffer. In vitro binding studies demonstrated that the intracellular segment of PTPmu binds directly to the intracellular domain of E-cadherin, but not to alpha- or beta-catenin. Consistent with their ability to interact in vivo, PTPmu, cadherins, and catenins all localized to points of cell-cell contact in MvLu cells, as assessed by immunocytochemical staining. After pervanadate treatment of MvLu cells, which inhibits cellular tyrosine phosphatase activity including PTPmu, the cadherins associated with PTPmu are now found in a tyrosine-phosphorylated form, indicating that the cadherins may be an endogenous substrate for PTPmu. These data suggest that PTPmu may be one of the enzymes that regulates the dynamic tyrosine phosphorylation, and thus function, of the cadherin/catenin complex in vivo.

scholarly journals Hyaluronan binding function of CD44 is transiently activated on T cells during an in vivo immune response.

1994 ◽  
Vol 180 (1) ◽  
pp. 383-387 ◽  
Author(s):  
J Lesley ◽  
N Howes ◽  
A Perschl ◽  
R Hyman
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Surface Receptor ◽  
Target Cells ◽  
Cd44 Isoforms ◽  
Effector Cells ◽  
Binding Function ◽  
Cytotoxic Effector

Though CD44 functions as a cell surface receptor for hyaluronan (HA) in some cell lines, most normal hematopoietic cells expressing CD44 do not bind HA. Certain CD44-specific monoclonal antibodies (mAbs) can rapidly induce CD44-mediated HA binding in normal murine T cells. This observation suggests that in vivo mechanisms may exist for activating the HA receptor function of CD44 on normal T cells. Here, it is shown that up to one third of splenic T cells are capable of CD44-mediated binding of fluorescein-conjugated HA (Fl-HA) during an in vivo allogeneic response. HA binding activity peaks at 7-8 d postinjection and declines rapidly. These rapid kinetics could be the result of transient activation of CD44 function and/or differentiation or expansion of short-lived population(s) that have constitutive HA-binding function. Both CD4 and CD8 T cells are included in the HA binding population which is strongly CD44 positive. After separation of HA-binding cells from nonbinding cells by cell sorting, it is shown that almost all cytotoxic effector cells are found in the HA-binding population. However, there is no evidence that CD44-mediated HA recognition is directly involved in the killing of target cells, since cytotoxicity could not be inhibited by CD44-specific mAbs that inhibit HA binding or by soluble HA. PCR amplification of cDNA reverse transcribed from RNA of sorted HA-binding cells indicated no evidence for CD44 isoforms other than the standard (hematopoietic) form. Though CD44 expression is known to be elevated upon T cell activation, and, as shown here, HA-binding function is induced in a portion of CD44-expressing T cells including cytotoxic effector cells, the role of CD44 and HA-recognition in immune responses is not known.

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