Thrombomucin, a novel cell surface protein that defines thrombocytes and multipotent hematopoietic progenitors

Abstract

MEP21 is an avian antigen specifically expressed on the surface of Myb-Ets-transformed multipotent hematopoietic precursors (MEPs) and of normal thrombocytes. Using nanoelectrospray tandem mass spectrometry, we have sequenced and subsequently cloned the MEP21 cDNA and named the gene thrombomucin as it encodes a 571-amino acid protein with an extracellular domain typical of the mucin family of proteoglycans. Thrombomucin is distantly related to CD34, the best characterized and most used human hematopoietic stem cell marker. It is also highly homologous in its transmembrane/intracellular domain to podocalyxinlike protein-1, a rabbit cell surface glycoprotein of kidney podocytes. Single cell analysis of yolk sac cells from 3-d-old chick embryos revealed that thrombomucin is expressed on the surface of both lineage-restricted and multipotent progenitors. In the bone marrow, thrombomucin is also expressed on mono- and multipotent progenitors, showing an overlapping but distinct expression pattern from that of the receptor-type stem cell marker c-kit. These observations strengthen the notion that the Myb-Ets oncoprotein can induce the proliferation of thrombomucin-positive hematopoietic progenitors that have retained the capacity to differentiate along multiple lineages. They also suggest that thrombomucin and CD34 form a family of stem cell-specific proteins with possibly overlapping functions in early hematopoietic progenitors.

Figure 1

Expression of MEP21 protein by peripheral blood thrombocytes. (A and B) Immunofluorescence analysis of peripheral blood leukocytes from 5-wk-old chicks. (A) Cells were stained with a mouse mAb to α_IIbβ₃ integrin followed by a phycoerythrin-conjugated anti–mouse antibody and an FITC-coupled MEP21 antibody. (B) Another aliquot of the above cells was stained with MEP21 antibody followed by a phycoerythrin-conjugated anti–mouse antibody. MEP21+ and MEP21− fractions (gates R1 and R2, respectively) were sorted by flow cytometry and gave populations of >98% purity. (C) DiffQuik stained cells from R1 and R2 fractions. (D) Western blot analysis of MEP21 expression of hematopoietic cell lines and sorted peripheral blood cells. Designations of cells are indicated on the top of each lane.

Figure 1

Expression of MEP21 protein by peripheral blood thrombocytes. (A and B) Immunofluorescence analysis of peripheral blood leukocytes from 5-wk-old chicks. (A) Cells were stained with a mouse mAb to α_IIbβ₃ integrin followed by a phycoerythrin-conjugated anti–mouse antibody and an FITC-coupled MEP21 antibody. (B) Another aliquot of the above cells was stained with MEP21 antibody followed by a phycoerythrin-conjugated anti–mouse antibody. MEP21+ and MEP21− fractions (gates R1 and R2, respectively) were sorted by flow cytometry and gave populations of >98% purity. (C) DiffQuik stained cells from R1 and R2 fractions. (D) Western blot analysis of MEP21 expression of hematopoietic cell lines and sorted peripheral blood cells. Designations of cells are indicated on the top of each lane.

Figure 1

Expression of MEP21 protein by peripheral blood thrombocytes. (A and B) Immunofluorescence analysis of peripheral blood leukocytes from 5-wk-old chicks. (A) Cells were stained with a mouse mAb to α_IIbβ₃ integrin followed by a phycoerythrin-conjugated anti–mouse antibody and an FITC-coupled MEP21 antibody. (B) Another aliquot of the above cells was stained with MEP21 antibody followed by a phycoerythrin-conjugated anti–mouse antibody. MEP21+ and MEP21− fractions (gates R1 and R2, respectively) were sorted by flow cytometry and gave populations of >98% purity. (C) DiffQuik stained cells from R1 and R2 fractions. (D) Western blot analysis of MEP21 expression of hematopoietic cell lines and sorted peripheral blood cells. Designations of cells are indicated on the top of each lane.

Figure 2

Sequencing of MEP21 by nanoelectrospray mass spectrometry. (A) Mass spectrum of the unseperated peptide mixture obtained after in-gel tryptic digestion of the protein band. Tryptic peptide ions of MEP21 are marked by the letter T and their charge state (number of protons attached). Peaks of trypsin autolysis products are designated by asterisks. (B) Peptide ion T₄ (A) was isolated and fragmented in the collision chamber of the mass spectrometer, leading to the spectrum shown. Fragmentation of tryptic peptides predominantly produces nested sets of fragments containing the peptide COOH terminus (Y″ ₁, Y″ _2″, etc. [Roepstorff and Fohlmann, 1984]), which allow assignment of the sequence by their mass differences. (C) Tandem mass spectrum of the same peptide as in B after esterification of the whole peptide mixture. Esterification results in 14-D mass shifts for the ions containing COOH terminus plus an additional shift of 14 D for each Asp and Glu residue as indicated by filled circles. Comparison of the tandem mass spectra of native and esterified peptide, B and C, allowed unambiguous assignment of the peptide sequence. Note that the isobaric amino acids Leu and Ile could not be distinguished and are designated by the letter L. The following peptide sequences were determined: T ₁, [AS] NEAFFEVFCSGR; T ₂, [AS] NEAFFEVFCSGRR; T ₃, WAVHVLVHR; T ₄, VLDPAAVFEELK; T ₅, VLDPAAVFEELKEK; T ₆, VLDPAAVFEELKEKR; T ₇, ALLFLNR.

Figure 3

Structure and coding capacity of MEP21 cDNA clones. (A) Schematic representation of MEP21 type 1, 2, and 3 cDNA clones. Solid lines, 5′- and 3′-untranslated regions; stippled lines, alternative 3′-untranslated region; and boxes, coding regions. PAS, polyadenylation signal; SP, signal peptide; TM, transmembrane region; S-T-P, serine/threonine/proline rich domain; C-C, potential disulfide-bonded domain. (B) Amino acid sequence alignment of MEP21 (derived from type 1 clone nucleotide sequence) and PCLP-1. Shaded boxes, identity; dots, gaps; asterisks, potential phosphorylation sites (see text); MEP21-1,2,4, peptides sequenced by mass spectroscopy; and TM, the putative transmembrane domain. (C) COOH-terminal amino acid alignments between MEP21 type 1, 2, and 3 clones. Vertical lines, sequence identity; and dots, gaps. These sequence data are available from GenBank/ EMBL/DDBJ under accession numbers Y13976, Y13977, and Y13978.

Figure 4

Endogenous and exogenous expression of MEP21 in hematopoietic cell lines. (A) Northern blot analysis of various hematopoietic cell lines using an MEP21 probe: erythroid lines HD3 and HD37; MEP cell lines HD57 and HD100; myeloblast line HD57M; macrophage line HD11; promyelocyte line HD13; eosinophil line 1A1; B cell line RP-12; T cell lines MSB1 and NPB4. (B) Western blot analysis of ectopically expressed MEP21 protein. Cells of the HD3 erythroid cell line were transfected with an expression vector containing MEP21 cDNA in the sense or antisense orientation plus the neo gene and selected for G418 resistance. Cl3 and Cl4, sense; and Cl2, antisense transfected cells.

Figure 5

Immunohistologic analysis of MEP21-reactive cells in 5-d-old chicken tissues. Arrows indicate podocytes in kidney; arteries, and capillaries in lung; capillaries in gut villi; and penicillary arteries and thrombocytes in spleen.

Figure 6

Embryonic expression of thrombomucin. 4-d-old chick embryos were analyzed for MEP21 expression by either whole-mount in situ staining (A) or staining of sections (B). Lower panels are high power magnification of upper panels. a, dorsal aorta; n, neural tube; g, glomerulus; d, duodenum; l, liver; v, heart ventricle; at, heart atrium. Arrow, intra-aortic hematopoietic cells.

Figure 7

FACS^® analysis, sorting, and colony assays of day three yolk sac cells. (A) A pool of embryonic day three yolk sac cells were stained with MEP21 antibody followed by phycoerythrin-coupled anti–mouse antibody and an FITC-coupled antibody to the erythroid marker JS4 and analyzed by FACS^®. (B) Cells from fractions R1 and R2 were sorted and 500 cells each plated in plasma clot for 6 d before analysis of colonies formed. Columns and error bars indicate the average colony numbers obtained per culture (with standard error) of three cultures. LE, late erythroid colonies; EE, early erythroid colonies; T/E, mixed thrombocyte erythroid colonies; T, thrombocytic colonies; M, myelomonocytic colonies; and Eo, eosinophilic colonies.

Figure 7

FACS^® analysis, sorting, and colony assays of day three yolk sac cells. (A) A pool of embryonic day three yolk sac cells were stained with MEP21 antibody followed by phycoerythrin-coupled anti–mouse antibody and an FITC-coupled antibody to the erythroid marker JS4 and analyzed by FACS^®. (B) Cells from fractions R1 and R2 were sorted and 500 cells each plated in plasma clot for 6 d before analysis of colonies formed. Columns and error bars indicate the average colony numbers obtained per culture (with standard error) of three cultures. LE, late erythroid colonies; EE, early erythroid colonies; T/E, mixed thrombocyte erythroid colonies; T, thrombocytic colonies; M, myelomonocytic colonies; and Eo, eosinophilic colonies.

Figure 8

Thrombomucin expression in multipotent progenitors of day three yolk sac. A total of 2,112 MEP21-positive cells (Fig. 7 A, R1) were sorted, seeded singly into 96-well plates, cultured for 6 d and colonies were classified by microscopic inspection. (A) Pie diagram illustrating the relative frequencies of the different colony types obtained: T, 177 thrombocytic; E, 128 erythroid; M, 119 myeloid; Eos, 3 eosinophilic; T/E, 23 thrombocytic/erythroid; M/T, 3 myelomonocytic/thrombocytic; and M/T/E, 4 myelomonocytic/thrombocytic/erythroid. (B) Light micrographs of two separate colonies containing macrophages, erythrocytes, and thrombocytes.

Figure 8

Thrombomucin expression in multipotent progenitors of day three yolk sac. A total of 2,112 MEP21-positive cells (Fig. 7 A, R1) were sorted, seeded singly into 96-well plates, cultured for 6 d and colonies were classified by microscopic inspection. (A) Pie diagram illustrating the relative frequencies of the different colony types obtained: T, 177 thrombocytic; E, 128 erythroid; M, 119 myeloid; Eos, 3 eosinophilic; T/E, 23 thrombocytic/erythroid; M/T, 3 myelomonocytic/thrombocytic; and M/T/E, 4 myelomonocytic/thrombocytic/erythroid. (B) Light micrographs of two separate colonies containing macrophages, erythrocytes, and thrombocytes.

Figure 9

FACS^® analysis, sorting, and colony assays of bone marrow cells. (A) Two-color immunofluorescence analysis of 5-d chick bone marrow. Cells were stained with MEP21 antibody followed by FITC-coupled anti–mouse antibody, biotinylated anti– c-kit antibodies and phycoerythrin-coupled streptavidin and analyzed by FACS^®. (B) Cells from fractions R1, R2, and R3 were sorted, 500 cells per plate seeded in plasma clots supplemented with either SCF plus anemic serum (black), SCF plus cMGF (red), or all three factors (white), and then cultured for 6 d before analysis. Columns and error bars indicate the average and standard deviation of two separate experiments. Colony designations are as in Fig. 6; BFU-E, burst forming unit erythroid, the earliest detectable erythroid precursors.

Figure 10

Micrographs of primary and secondary colonies obtained from bone marrow–derived progenitors. (A) Primary blast-type colony, 10 d after seeding of MEP21-positive cells (fraction R2) in medium containing SCF, anemic serum, and erythropoietin. (B and C) Secondary plasma clot colonies obtained after seeding two different blast colonies into plasma clot for 5 d and staining with DiffQuik. M, macrophages; E, erythrocytes; T, thrombocytes.

Figure 11

Differentiation potential and expression of thrombomucin by hematopoietic cells. (A) E26-transformed hematopoietic cells and (B) normal bone marrow cells. MEP, Myb-Ets– transformed progenitors; MYE, myelobasts; THR, thrombocytes; ERY, erythrocytes; EOS, eosinophils; GRA, granulocytes; MAC, macrophages. The broken arrow lines in A indicate that differentiation is normally blocked at the MEP stage, but is inducible (see text). The solid arrow lines in B denote the existence of multi- and monopotent progenitors as inferred from colony assays. Cell types expressing exclusively thrombomucin are indicated by the pink shaded areas surrounded by the solid lines; those expressing exclusively c-kit by the blue shaded area surrounded by the stippled line. Cell types expressing both antigens are indicated by the gray shaded areas.