scholarly journals Cell Surface Processing of CD109 by Meprin β Leads to the Release of Soluble Fragments and Reduced Expression on Extracellular Vesicles

2021 ◽  
Vol 9 ◽  
Author(s):  
Wiebke Lückstädt ◽  
Simon Bub ◽  
Tomas Koudelka ◽  
Egor Pavlenko ◽  
Florian Peters ◽  
...  
Keyword(s):  
Cell Surface ◽  
Extracellular Vesicles ◽  
Structural Model ◽  
Molecular Model ◽  
Particle Analysis ◽  
Release Mechanism ◽  
Cleavage Sites ◽  
Structural Localization ◽  
Hematopoietic Stem ◽  
Bait Region

Cluster of differentiation 109 (CD109) is a glycosylphosphatidylinositol (GPI)-anchored protein expressed on primitive hematopoietic stem cells, activated platelets, CD4+ and CD8+ T cells, and keratinocytes. In recent years, CD109 was also associated with different tumor entities and identified as a possible future diagnostic marker linked to reduced patient survival. Also, different cell signaling pathways were proposed as targets for CD109 interference including the TGFβ, JAK-STAT3, YAP/TAZ, and EGFR/AKT/mTOR pathways. Here, we identify the metalloproteinase meprin β to cleave CD109 at the cell surface and thereby induce the release of cleavage fragments of different size. Major cleavage was identified within the bait region of CD109 residing in the middle of the protein. To identify the structural localization of the bait region, homology modeling and single-particle analysis were applied, resulting in a molecular model of membrane-associated CD109, which allows for the localization of the newly identified cleavage sites for meprin β and the previously published cleavage sites for the metalloproteinase bone morphogenetic protein-1 (BMP-1). Full-length CD109 localized on extracellular vesicles (EVs) was also identified as a release mechanism, and we can show that proteolytic cleavage of CD109 at the cell surface reduces the amount of CD109 sorted to EVs. In summary, we identified meprin β as the first membrane-bound protease to cleave CD109 within the bait region, provide a first structural model for CD109, and show that cell surface proteolysis correlates negatively with CD109 released on EVs.

scholarly journals Flexibility of the Adenovirus Fiber Is Required for Efficient Receptor Interaction

2003 ◽  
Vol 77 (13) ◽  
pp. 7225-7235 ◽  
Author(s):  
Eugene Wu ◽  
Lars Pache ◽  
Dan J. Von Seggern ◽  
Tina-Marie Mullen ◽  
Yeshi Mikyas ◽  
...  
Keyword(s):  
Virus Infection ◽  
Cell Surface ◽  
Cell Attachment ◽  
Molecular Model ◽  
Host Cells ◽  
Receptor Interaction ◽  
Particle Analysis ◽  
Fiber Protein ◽  
Fiber Proteins ◽  
Fiber Flexibility

ABSTRACT The adenovirus (Ad) fiber protein mediates Ad binding to the coxsackievirus and Ad receptor (CAR) and is thus a major determinant of viral tropism. The fiber contains three domains: an N-terminal tail that anchors the fiber to the viral capsid, a central shaft region of variable length and flexibility, and a C-terminal knob domain that binds to cell receptors. Ad type 37 (Ad37), a subgroup D virus associated with severe ocular infections, is unable to use CAR efficiently to infect host cells, despite containing a CAR binding site in its fiber knob. We hypothesized that the relatively short, inflexible Ad37 fiber protein restricts interactions with CAR at the cell surface. To test this hypothesis, we analyzed the infectivity and binding of recombinant Ad particles containing modified Ad37 or Ad5 fiber proteins. Ad5 particles equipped with a truncated Ad5 fiber or with a chimeric fiber protein comprised of the Ad5 knob fused to the short, rigid Ad37 shaft domain had significantly reduced infectivity and attachment. In contrast, placing the Ad37 knob onto the long, flexible Ad5 shaft allowed CAR-dependent virus infection and cell attachment, demonstrating the importance of the shaft domain in receptor usage. Increasing fiber rigidity by substituting the predicted flexibility modules in the Ad5 shaft with the corresponding regions of the rigid Ad37 fiber dramatically reduced both virus infection and cell attachment. Cryoelectron microscopy (cryo-EM) single-particle analysis demonstrated the increased rigidity of this chimeric fiber. These studies demonstrate that both length and flexibility of the fiber shaft regulate CAR interaction and provide a molecular explanation for the use of alternative receptors by subgroup D Ad with ocular tropism. We present a molecular model for Ad-CAR interactions at the cell surface that explains the significance of fiber flexibility in cell attachment.

scholarly journals Interplay between LPA2 and LPA3 in LPA-mediated phosphatidylserine cell surface exposure and extracellular vesicles release by erythrocytes

2021 ◽  
pp. 114667
Author(s):  
Stephan Hasse ◽  
Anne-Claire Duchez ◽  
Paul Fortin ◽  
Eric Boilard ◽  
Sylvain G. Bourgoin
Keyword(s):  
Cell Surface ◽  
Extracellular Vesicles

scholarly journals Isolation of small, primitive human hematopoietic stem cells: distribution of cell surface cytokine receptors and growth in SCID-Hu mice

Blood ◽  
1995 ◽  
Vol 86 (2) ◽  
pp. 512-523 ◽  
Author(s):  
JE Wagner ◽  
D Collins ◽  
S Fuller ◽  
LR Schain ◽  
AE Berson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Surface ◽  
Cell Population ◽  
Size Fraction ◽  
Cell Surface Antigens ◽  
Inhibitory Protein ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cell Fractions

Human CD34+ cells were subfractionated into three size classes using counterflow centrifugal elutriation followed by immunoadsorption to polystyrene cell separation devices. The three CD34+ cell fractions (Fr), Fr 25/29, Fr 33/37, and Fr RO, had mean sizes of 8.5, 9.3 and 13.5 microns, respectively. The majority of cells in the large Fr RO CD34+ cell population expressed the committed stage antigens CD33, CD19, CD38, or HLA-DR and contained the majority of granulocyte- macrophage colony-forming units (CFU-GM), burst-forming units-erythroid (BFU-E), and CFU-mixed lineage (GEMM). In contrast, the small Fr 25/29 CD34+ cells were devoid of committed cell surface antigens and lacked colony-forming activity. When seeded to allogeneic stroma, Fr RO CD34+ cells produced few CFU-GM at week 5, whereas cells from the Fr 25/29 CD34+ cell population showed a 30- to 55-fold expansion of myeloid progenitors at this same time point. Furthermore, CD34+ cells from each size fraction supported ontogeny of T cells in human thymus/liver grafts in severe combined immunodeficient (SCID) mice. Upon cell cycle analyses, greater than 97% of the Fr 25/29 CD34+ cells were in G0/G1 phase, whereas greater proportions of the two larger CD34+ cell fractions were in active cell cycle. Binding of the cytokines interleukin (IL)-1 alpha, IL-3, IL-6, stem cell factor (SCF), macrophage inhibitory protein (MIP)-1 alpha, granulocyte colony- stimulating factor (G-CSF), and granulocyte-macrophage (GM)-CSF to these CD34+ cell populations was also analyzed by flow cytometry. As compared with the larger CD34+ cell fractions, cells in the small Fr 25/29 CD34+ cell population possessed the highest numbers of receptors for SCF, MIP1 alpha, and IL-1 alpha. Collectively, these results indicate that the Fr 25/29 CD34+ cell is a very primitive, quiescent progenitor cell population possessing a high number of receptors for SCF and MIP1 alpha and capable of yielding both myeloid and lymphoid lineages when placed in appropriate in vitro or in vivo culture conditions.

scholarly journals The Power of Extracellular Vesicles in Myeloproliferative Neoplasms: “Crafting” a Microenvironment That Matters

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2316
Author(s):  
Lucia Catani ◽  
Michele Cavo ◽  
Francesca Palandri
Keyword(s):  
Extracellular Vesicles ◽  
Immune Escape ◽  
Myeloproliferative Neoplasms ◽  
Cell Communication ◽  
Bioactive Molecules ◽  
Driver Genes ◽  
Hematopoietic Stem ◽  
Increased Risk ◽  
Patients Experience

Myeloproliferative Neoplasms (MPN) are acquired clonal disorders of the hematopoietic stem cells and include Essential Thrombocythemia, Polycythemia Vera and Myelofibrosis. MPN are characterized by mutations in three driver genes (JAK2, CALR and MPL) and by a state of chronic inflammation. Notably, MPN patients experience increased risk of thrombosis, disease progression, second neoplasia and evolution to acute leukemia. Extracellular vesicles (EVs) are a heterogeneous population of microparticles with a role in cell-cell communication. The EV-mediated cross-talk occurs via the trafficking of bioactive molecules such as nucleic acids, proteins, metabolites and lipids. Growing interest is focused on EVs and their potential impact on the regulation of blood cancers. Overall, EVs have been suggested to orchestrate the complex interplay between tumor cells and the microenvironment with a pivotal role in “education” and “crafting” of the microenvironment by regulating angiogenesis, coagulation, immune escape and drug resistance of tumors. This review is focused on the role of EVs in MPN. Specifically, we will provide an overview of recent findings on the involvement of EVs in MPN pathogenesis and discuss opportunities for their potential application as diagnostic and prognostic biomarkers.

Functional analysis and molecular model of the human urate transporter/channel, hUAT

2002 ◽  
Vol 283 (1) ◽  
pp. F150-F163 ◽  
Author(s):  
Edgar Leal-Pinto ◽  
B. Eleazar Cohen ◽  
Michael S. Lipkowitz ◽  
Ruth G. Abramson
Keyword(s):  
Functional Analysis ◽  
Lipid Bilayers ◽  
Structural Model ◽  
Transmembrane Domain ◽  
Single Channel ◽  
Molecular Model ◽  
Open Probability ◽  
Homologous Proteins ◽  
Urate Transporter ◽  
Binding Domains

Recombinant protein, designated hUAT, the human homologue of the rat urate transporter/channel (UAT), functions as a highly selective urate channel in lipid bilayers. Functional analysis indicates that hUAT activity, like UAT, is selectively blocked by oxonate from its cytosolic side, whereas pyrazinoate and adenosine selectively block from the channel's extracellular face. Importantly, hUAT is a galectin, a protein with two β-galactoside binding domains that bind lactose. Lactose significantly increased hUAT open probability but only when added to the channel's extracellular side. This effect on open probability was mimicked by glucose, but not ribose, suggesting a role for extracellular glucose in regulating hUAT channel activity. These functional observations support a four-transmembrane-domain structural model of hUAT, as previously predicted from the primary structure of UAT. hUAT and UAT, however, are not functionally identical: hUAT has a significantly lower single-channel conductance and open probability is voltage independent. These differences suggest that evolutionary changes in specific amino acids in these highly homologous proteins are functionally relevant in defining these biophysical properties.

scholarly journals Phenotype analysis of hematopoietic CD34+ cell populations derived from human umbilical cord blood using flow cytometry and cDNA-polymerase chain reaction

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2103-2114 ◽  
Author(s):  
SJ Thoma ◽  
CP Lamping ◽  
BL Ziegler
Keyword(s):  
Flow Cytometry ◽  
Polymerase Chain Reaction ◽  
Cell Surface ◽  
Chain Reaction ◽  
Hematopoietic Stem ◽  
Hla Dr ◽  
Cd34 Cells ◽  
Rare Populations ◽  
Pcr Method ◽  
Polymerase Chain

Abstract A strategy to phenotype rare populations of hematopoietic cells expressing the cell-surface marker CD34 was studied. The antigenic phenotype of umbilical core blood (CB) CD34+ cells was investigated using flow cytometry and compared with the mRNA-phenotype determined by cDNA-polymerase chain reaction (cDNA-PCR) analysis. The cDNA-PCR method allowed an mRNA evaluation of small numbers of cells. Monoclonal antibodies and oligonucleotide primers that recognize myeloid, lymphoid, erythroid and platelet/megakaryocytic cell membrane antigens or corresponding mRNA transcripts were used. Evaluation by flow cytometry showed that the vast majority of CD34+ CB cells coexpressed CD38, CD18, HLA-DR, and CD33. Rare subpopulations of CD34+CD38-, CD34+CD18-, CD34+HLA-DR-, and CD34+CD33- were also identified. A large proportion of CD34+ CB cells expressed CD13, CD45R, and to a lesser extent CD71. The CD36, CD51, and CD61 antigens were identified on a small number of CD34+ cells. The three-color flow cytometry analysis showed that CD34+ cells stained with antibodies to CD61 and CD36 or CD51 can be divided into subsets that may represent progenitor cells committed to the erythroid and/or megakaryocytic lineage. A variety of other lineage-specific cell-surface antigens including pre-T-cell marker CD7 and markers of early B cells, ie, CD10 and CD19, were not coexpressed with CD34+. Using the cDNA-PCR it was seen that the mRNA phenotype of a small number of sorted CD34+ cells (purity > 98%) was negative for the markers CD2, CD14, CD16, CD20, CD21, CD22, CD41b, and glycophorin A that are expressed on differentiated cells but positive for CD34, CD7, CD19, CD36, and CD61. The results suggest that circulating CD34+CD7+ and CD34+CD19+ CB cells cannot be distinguished by flow cytometry but can be detected by cDNA-PCR. This indicates that CB either contains very low numbers of these progenitors or that the antigen density of CD7 and CD19 on CD34+ cells is below the detection limit of the flow cytometer. In contrast to flow cytometry, cDNA-PCR allows the phenotypic analysis of cells even if their number is small. Thus, the cDNA-PCR method can be useful in linking phenotype analyses, ie, markers of differentiation, to studies on gene expression within rare populations of hematopoietic stem cells.

scholarly journals Blood extracellular vesicles from healthy individuals regulate hematopoietic stem cells as humans age

Aging Cell ◽  
2020 ◽  
Vol 19 (11) ◽  
Author(s):  
Isabelle Grenier‐Pleau ◽  
Kathrin Tyryshkin ◽  
Tri Dung Le ◽  
John Rudan ◽  
Eric Bonneil ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Extracellular Vesicles ◽  
Healthy Individuals ◽  
Hematopoietic Stem
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