Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

AbuSamra, Dina B.; Al-Kilani, Alia; Hamdan, Samir M.; Sakashita, Kosuke; Gadhoum, Samah Zeineb; Merzaban, Jasmeen S.

doi:10.1074/jbc.m114.629451

Cited by 28 publications

(63 citation statements)

References 54 publications

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“…Captured proteins were recovered and then subjected to western blot analysis as detailed in supplemental Materials and methods. Immunostaining with CD34-mAb verified the presence of CD34 ( Figure 3A inset), whereas immunostaining for the presence of PSGL-1, a highly expressed E-selL on hematopoietic cells, 33,34,36 verified its absence (Figure 3A inset). As estimated from Equation 1 in supplemental Materials and methods, we observed low ligand-capturing efficiency, likely attributed to the immobilization of the mAb rendering its binding site inaccessible, with only 9.5% and 20.2% for CD34 and CD44, respectively.…”

Section: Cd34 Is An E-sell Under Flow Conditionsmentioning

confidence: 99%

“…Following the injection of the HSPC-enriched lysate, significant RU accumulated in the CD34 and CD44 flow cells, whereas only residual RUs were detected in the control flow cells that were either left blank (no mAb) or were immobilized with isotype control (MsIgG) ( Figure 3A). It should be noted that the CD44 mAb used here captured a mixture of CD44 glycoforms 36 of which only a fraction bound E-selectin. Furthermore, the purity of the captured CD34 was assessed as described previously.…”

Section: Cd34 Is An E-sell Under Flow Conditionsmentioning

confidence: 99%

“…To examine whether native CD34 on human HSPC-enriched populations displayed functional E-selL activity in flow-based assays, we employed 3 approaches: the SPR-based binding assay developed in our laboratory, 36 the Stamper-Woodruff assay, 37 and the blot-rolling assay. 38,39 We compared CD34 binding to the wellestablished E-selL, CD44 (ie, HCELL).…”

Section: Cd34 Is An E-sell Under Flow Conditionsmentioning

confidence: 99%

“…We evaluated the efficiency of neuraminidase to digest sialic acid using western blots and probing with the QBend-10 antibody (Class 2 mAb) to track the shift in the MW of CD34 from ;120 kDa to 150 kDa ( Figure 6B lower panels). 36,41 To determine whether the E-selectin binding epitope resides on N-and/or O-glycans, we treated the KG1a lysate with PNGase F or OSGE, respectively. We found that removing the N-glycans resulted in only a small reduction in RU max (1.4-fold) and a similar dissociation constant (k off-apparent ) as the control lysate ( Figure 6C).…”

Section: Cd34 Binding To E-selectin Is Dependent On Sialofucosylated mentioning

confidence: 99%

“…Furthermore, the purity of the captured CD34 was assessed as described previously. 36 Briefly, following lysate injection, a 5-minute bufferwashing step was introduced in order to eliminate potential multiligand complexes. Captured proteins were recovered and then subjected to western blot analysis as detailed in supplemental Materials and methods.…”

Section: Cd34 Is An E-sell Under Flow Conditionsmentioning

confidence: 99%

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Not just a marker: CD34 on human hematopoietic stem/progenitor cells dominates vascular selectin binding along with CD44

AbuSamra¹,

Aleisa²,

Al-Amoodi³

et al. 2017

Blood Advances

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Section: Cd34 Is An E-sell Under Flow Conditionsmentioning

confidence: 99%

Section: Cd34 Is An E-sell Under Flow Conditionsmentioning

confidence: 99%

Section: Cd34 Is An E-sell Under Flow Conditionsmentioning

confidence: 99%

Section: Cd34 Binding To E-selectin Is Dependent On Sialofucosylated mentioning

confidence: 99%

Section: Cd34 Is An E-sell Under Flow Conditionsmentioning

confidence: 99%

See 3 more Smart Citations

Not just a marker: CD34 on human hematopoietic stem/progenitor cells dominates vascular selectin binding along with CD44

AbuSamra¹,

Aleisa²,

Al-Amoodi³

et al. 2017

Blood Advances

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Immunological Adhesion and Homing Molecules

Baldanzi

2019

Encyclopedia of Life Sciences

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The immunosurveillance function of leukocytes requires a strictly controlled trafficking between lymphoid and nonlymphoid tissues. Thus, white blood cells are continuously scanning the body for sign of infection or damage with lineage‐specific pathways. Triggered by chemokines and other danger signals, those movements require selective interactions mediated by adhesion and homing molecules expressed by immune and endothelial cells. Typically, the transendothelial migration is initiated by specific interactions between selectins and glycosylated proteins, which allow the initial tethering and rolling of leukocytes on endothelial cells. In the presence of chemokines, rolling is followed by firm arrest mediated by active integrins binding to immunoglobulin superfamily ligands and culminates with extravasation. The expression pattern and structural features of those adhesion molecules dictated the cell‐specific homing governing the immune response. Key Concepts Leukocytes exit from circulation to home at secondary lymphoid organs and peripheral tissues. Essential steps of extravasation are tethering, rolling, firm adhesion and diapedesis. The typical homing cascade sees selectins binding to glycoproteins during rolling, followed by chemokine mediated integrin activation promoting firm adhesion. Endothelial cells have an active role in lymphocyte recruitment, presentation of chemokines and transendothelial transport of leukocytes. Homing molecules are controlled by the engagement by ligands and by the shear force applied to them.

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Correlation between plasma PSGL‐1 and FIGO stage, tumor metastasis, and survival in epithelial ovarian cancer

Li,

Fang,

Gao

et al. 2024

Biotech and App Biochem

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Plasma circulating P‐selectin glycoprotein ligand‐1 (PSGL‐1) levels and its clinical correlation in patients with epithelial ovarian cancer (EOC) are unknown. The study determined plasma PSGL‐1 levels in EOC patients and investigated its relationship with clinicopathological factors and prognosis. Plasma PSGL‐1 levels were measured using ELISA in 69 patients with EOC, 34 patients with benign ovarian cystadenoma, and 36 healthy controls. Subsequently, the relationship between PSGL‐1 levels and clinicopathological characteristics of patients, as well as the prognosis of EOC patients, was examined. Additionally, the specificity and sensitivity of plasma PSGL‐1 were assessed through ROC curve analysis. Plasma PSGL‐1 was upregulated in EOC patients compared with healthy subjects and/or patients with benign ovarian cystadenoma (p < 0.01). Elevated levels of PSGL‐1 in the plasma were positively associated with advanced FIGO stage (p < 0.001), tumor size (p = 0.001), tumor metastasis (p = 0.036), and tumor recurrence (p = 0.013), while was negatively correlated with residual tumor size (p < 0.001). Kaplan–Meier survival analysis showed that high plasma PSGL‐1 levels were associated with progression‐free survival (p = 0.0345). In univariate and multivariate Cox regression analyses, PSGL‐1 (HR = 1.456, p = 0.009) was an independent prognostic marker. Plasma PSGL‐1 levels distinguished EOC patients and healthy individuals (AUC = 0.905), patients at late and early FIGO stages (AUC = 0.886), and metastatic and non‐metastatic EOC (AUC = 0.722). The expression of plasma PSGL‐1 is significantly increased in patients with EOC, serving as a reliable biomarker to differentiate between healthy individuals and those with EOC. Furthermore, PSGL‐1 in patients is correlated with prognostic indicators, such as advanced FIGO stage, tumor lymph node metastasis, and progression‐free survival.

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Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

Cited by 28 publications

References 54 publications

Not just a marker: CD34 on human hematopoietic stem/progenitor cells dominates vascular selectin binding along with CD44

Not just a marker: CD34 on human hematopoietic stem/progenitor cells dominates vascular selectin binding along with CD44

Immunological Adhesion and Homing Molecules

Correlation between plasma PSGL‐1 and FIGO stage, tumor metastasis, and survival in epithelial ovarian cancer

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