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CD44 is a major E-selectin ligand on human hematopoietic progenitor cells.

Dimitroff CJ, Lee JY, Rafii S, Fuhlbrigge RC, Sackstein R - J. Cell Biol. (2001)

Bottom Line: The E-selectin ligand activity of CD44 is conferred by the elaboration of sialylated, fucosylated binding determinants on N-glycans.This glycoform of CD44 is expressed on primitive CD34+ human HPCs, but not on more mature hematopoietic cells.These findings offer new insights into the structural biology and physiology of CD44, and into the molecular basis of E-selectin-dependent adhesive interactions that direct homing of human HPC to BM.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

ABSTRACT
E-selectin plays a critical role in mediating tissue-specific homing of T cells into skin, and of primitive hematopoietic progenitor cells (HPCs) into bone marrow (BM). Though it is known that a glycoform of PSGL-1 (CLA) functions as the principal E-selectin ligand on human T lymphocytes, the E-selectin ligand(s) of human HPCs has not been identified. We used a shear-based adherence assay to analyze and define the E-selectin ligand activity of membrane proteins from human HPCs. Our data show that PSGL-1 expressed on human HPCs is an E-selectin ligand, and that HPCs also express a previously unrecognized E-selectin ligand, CD44. The E-selectin ligand activity of CD44 is conferred by the elaboration of sialylated, fucosylated binding determinants on N-glycans. This glycoform of CD44 is expressed on primitive CD34+ human HPCs, but not on more mature hematopoietic cells. Under physiologic flow conditions, this molecule mediates E-selectin-dependent rolling interactions over a wider shear range than that of PSGL-1, and promotes human HPC rolling interactions on E-selectin expressed on human BM endothelial cells. These findings offer new insights into the structural biology and physiology of CD44, and into the molecular basis of E-selectin-dependent adhesive interactions that direct homing of human HPC to BM.

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HECA-452–reactive CD44 from freshly isolated normal human HPCs and from human leukemic blasts functions as an E-selectin ligand. (Panel A) HECA-452 staining of CD44 immunoprecipitated from (a) human BM mononuclear cells (108 cells), (b) CD34−/lineage+ cells (107 cells), (c) CD34+/lineage− cells (107 cells), (d) CD34−/lineage+ cells (108 cells). HECA-452 staining of CD44 and CHO-E cell rolling was observable only on the 100-kD CD44 immunoprecipitated from CD34+/lineage− cells (data are mean ± SD cell rolling/field on the 100-kD band). (B) Membrane proteins (50 μg) isolated from circulating blasts from an AML (M5) were resolved on a 9% SDS-PAGE gel and immunoblotted with HECA-452. Treatment of membrane proteins with N-glycosidase F markedly diminished HECA-452 staining. (C) Blot rolling assay results of AML (M5) membrane protein (50 μg) immunoprecipitated with isotype control or with Hermes-1 mAb, and of N-glycosidase F–treated Hermes-1 immunoprecipitates. Though data shown in parentheses is CHO-E cell rolling over the 100-kD band, no rolling was observed over the entire length of the lane corresponding to N-glycosidase F–treated protein. (D) Immunoprecipitated CD44 from membrane preparations (50 μg) of an AML (M0), AML (M1) and atypical CML (brc/abl−), and of human BMEC line (BMEC-1; 100 μg total protein), was separated on a 6% SDS-PAGE gel, immunostained with HECA-452, and evaluated for E-selectin ligand activity. E-selectin ligand activity correlates with intensity of HECA-452 staining of 100-kD band. (E) Western blot of immunoprecipitated CD44 from BMEC-1 stained with Hermes-1 mAb.
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Figure 6: HECA-452–reactive CD44 from freshly isolated normal human HPCs and from human leukemic blasts functions as an E-selectin ligand. (Panel A) HECA-452 staining of CD44 immunoprecipitated from (a) human BM mononuclear cells (108 cells), (b) CD34−/lineage+ cells (107 cells), (c) CD34+/lineage− cells (107 cells), (d) CD34−/lineage+ cells (108 cells). HECA-452 staining of CD44 and CHO-E cell rolling was observable only on the 100-kD CD44 immunoprecipitated from CD34+/lineage− cells (data are mean ± SD cell rolling/field on the 100-kD band). (B) Membrane proteins (50 μg) isolated from circulating blasts from an AML (M5) were resolved on a 9% SDS-PAGE gel and immunoblotted with HECA-452. Treatment of membrane proteins with N-glycosidase F markedly diminished HECA-452 staining. (C) Blot rolling assay results of AML (M5) membrane protein (50 μg) immunoprecipitated with isotype control or with Hermes-1 mAb, and of N-glycosidase F–treated Hermes-1 immunoprecipitates. Though data shown in parentheses is CHO-E cell rolling over the 100-kD band, no rolling was observed over the entire length of the lane corresponding to N-glycosidase F–treated protein. (D) Immunoprecipitated CD44 from membrane preparations (50 μg) of an AML (M0), AML (M1) and atypical CML (brc/abl−), and of human BMEC line (BMEC-1; 100 μg total protein), was separated on a 6% SDS-PAGE gel, immunostained with HECA-452, and evaluated for E-selectin ligand activity. E-selectin ligand activity correlates with intensity of HECA-452 staining of 100-kD band. (E) Western blot of immunoprecipitated CD44 from BMEC-1 stained with Hermes-1 mAb.

Mentions: To determine whether CD44 naturally expressed on normal human HPCs functions as an E-selectin ligand, we investigated the distribution of HECA-452 reactivity and E-selectin ligand activity of CD44 expressed on early CD34+ cells and more mature (CD34−/lineage+) human BM cells (including populations enriched for monocytes [CD14+]), granulocytes [CD15+], and lymphocytes (B cells [CD19+] and T cells [CD3+]). Notably, though SDS-PAGE of Hermes-1 immunoprecipitates of KG1a cells reveals three bands (100, 120, and 190 kD), only a single 100-kD CD44 was immunoprecipitated from both CD34+ and lineage+/CD34− cells, and only CD44 from CD34+ cells stained with HECA-452 and functioned as an E-selectin ligand (Fig. 6 A). Identical results were obtained whether CD34+ cells were enriched by negative or positive selection. Indeed, even when tenfold excess lineage+ cell membrane protein was used for CD44 immunoprecipitation, there was still neither HECA-452 staining of CD44, nor E-selectin ligand activity of CD44 (Fig. 6 A). Moreover, immobilized on plastic, CD44 immunoprecipitated only from CD34+/CD44+ cells supported CHO-E cell rolling, whereas immunoprecipitated CD44 from CD34− cells did not possess any E-selectin ligand activity.


CD44 is a major E-selectin ligand on human hematopoietic progenitor cells.

Dimitroff CJ, Lee JY, Rafii S, Fuhlbrigge RC, Sackstein R - J. Cell Biol. (2001)

HECA-452–reactive CD44 from freshly isolated normal human HPCs and from human leukemic blasts functions as an E-selectin ligand. (Panel A) HECA-452 staining of CD44 immunoprecipitated from (a) human BM mononuclear cells (108 cells), (b) CD34−/lineage+ cells (107 cells), (c) CD34+/lineage− cells (107 cells), (d) CD34−/lineage+ cells (108 cells). HECA-452 staining of CD44 and CHO-E cell rolling was observable only on the 100-kD CD44 immunoprecipitated from CD34+/lineage− cells (data are mean ± SD cell rolling/field on the 100-kD band). (B) Membrane proteins (50 μg) isolated from circulating blasts from an AML (M5) were resolved on a 9% SDS-PAGE gel and immunoblotted with HECA-452. Treatment of membrane proteins with N-glycosidase F markedly diminished HECA-452 staining. (C) Blot rolling assay results of AML (M5) membrane protein (50 μg) immunoprecipitated with isotype control or with Hermes-1 mAb, and of N-glycosidase F–treated Hermes-1 immunoprecipitates. Though data shown in parentheses is CHO-E cell rolling over the 100-kD band, no rolling was observed over the entire length of the lane corresponding to N-glycosidase F–treated protein. (D) Immunoprecipitated CD44 from membrane preparations (50 μg) of an AML (M0), AML (M1) and atypical CML (brc/abl−), and of human BMEC line (BMEC-1; 100 μg total protein), was separated on a 6% SDS-PAGE gel, immunostained with HECA-452, and evaluated for E-selectin ligand activity. E-selectin ligand activity correlates with intensity of HECA-452 staining of 100-kD band. (E) Western blot of immunoprecipitated CD44 from BMEC-1 stained with Hermes-1 mAb.
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Figure 6: HECA-452–reactive CD44 from freshly isolated normal human HPCs and from human leukemic blasts functions as an E-selectin ligand. (Panel A) HECA-452 staining of CD44 immunoprecipitated from (a) human BM mononuclear cells (108 cells), (b) CD34−/lineage+ cells (107 cells), (c) CD34+/lineage− cells (107 cells), (d) CD34−/lineage+ cells (108 cells). HECA-452 staining of CD44 and CHO-E cell rolling was observable only on the 100-kD CD44 immunoprecipitated from CD34+/lineage− cells (data are mean ± SD cell rolling/field on the 100-kD band). (B) Membrane proteins (50 μg) isolated from circulating blasts from an AML (M5) were resolved on a 9% SDS-PAGE gel and immunoblotted with HECA-452. Treatment of membrane proteins with N-glycosidase F markedly diminished HECA-452 staining. (C) Blot rolling assay results of AML (M5) membrane protein (50 μg) immunoprecipitated with isotype control or with Hermes-1 mAb, and of N-glycosidase F–treated Hermes-1 immunoprecipitates. Though data shown in parentheses is CHO-E cell rolling over the 100-kD band, no rolling was observed over the entire length of the lane corresponding to N-glycosidase F–treated protein. (D) Immunoprecipitated CD44 from membrane preparations (50 μg) of an AML (M0), AML (M1) and atypical CML (brc/abl−), and of human BMEC line (BMEC-1; 100 μg total protein), was separated on a 6% SDS-PAGE gel, immunostained with HECA-452, and evaluated for E-selectin ligand activity. E-selectin ligand activity correlates with intensity of HECA-452 staining of 100-kD band. (E) Western blot of immunoprecipitated CD44 from BMEC-1 stained with Hermes-1 mAb.
Mentions: To determine whether CD44 naturally expressed on normal human HPCs functions as an E-selectin ligand, we investigated the distribution of HECA-452 reactivity and E-selectin ligand activity of CD44 expressed on early CD34+ cells and more mature (CD34−/lineage+) human BM cells (including populations enriched for monocytes [CD14+]), granulocytes [CD15+], and lymphocytes (B cells [CD19+] and T cells [CD3+]). Notably, though SDS-PAGE of Hermes-1 immunoprecipitates of KG1a cells reveals three bands (100, 120, and 190 kD), only a single 100-kD CD44 was immunoprecipitated from both CD34+ and lineage+/CD34− cells, and only CD44 from CD34+ cells stained with HECA-452 and functioned as an E-selectin ligand (Fig. 6 A). Identical results were obtained whether CD34+ cells were enriched by negative or positive selection. Indeed, even when tenfold excess lineage+ cell membrane protein was used for CD44 immunoprecipitation, there was still neither HECA-452 staining of CD44, nor E-selectin ligand activity of CD44 (Fig. 6 A). Moreover, immobilized on plastic, CD44 immunoprecipitated only from CD34+/CD44+ cells supported CHO-E cell rolling, whereas immunoprecipitated CD44 from CD34− cells did not possess any E-selectin ligand activity.

Bottom Line: The E-selectin ligand activity of CD44 is conferred by the elaboration of sialylated, fucosylated binding determinants on N-glycans.This glycoform of CD44 is expressed on primitive CD34+ human HPCs, but not on more mature hematopoietic cells.These findings offer new insights into the structural biology and physiology of CD44, and into the molecular basis of E-selectin-dependent adhesive interactions that direct homing of human HPC to BM.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

ABSTRACT
E-selectin plays a critical role in mediating tissue-specific homing of T cells into skin, and of primitive hematopoietic progenitor cells (HPCs) into bone marrow (BM). Though it is known that a glycoform of PSGL-1 (CLA) functions as the principal E-selectin ligand on human T lymphocytes, the E-selectin ligand(s) of human HPCs has not been identified. We used a shear-based adherence assay to analyze and define the E-selectin ligand activity of membrane proteins from human HPCs. Our data show that PSGL-1 expressed on human HPCs is an E-selectin ligand, and that HPCs also express a previously unrecognized E-selectin ligand, CD44. The E-selectin ligand activity of CD44 is conferred by the elaboration of sialylated, fucosylated binding determinants on N-glycans. This glycoform of CD44 is expressed on primitive CD34+ human HPCs, but not on more mature hematopoietic cells. Under physiologic flow conditions, this molecule mediates E-selectin-dependent rolling interactions over a wider shear range than that of PSGL-1, and promotes human HPC rolling interactions on E-selectin expressed on human BM endothelial cells. These findings offer new insights into the structural biology and physiology of CD44, and into the molecular basis of E-selectin-dependent adhesive interactions that direct homing of human HPC to BM.

Show MeSH
Related in: MedlinePlus