Marc Schapira

Evolutionary conservation of P-selectin glycoprotein ligand-1 primary structure and function.

BackgroundP-selectin glycoprotein ligand-1 (PSGL-1) plays a critical role in recruiting leukocytes in inflammatory lesions by mediating leukocyte rolling on selectins. Core-2 O-glycosylation of a N-terminal threonine and sulfation of at least one tyrosine residue of PSGL-1 are required for L- and P-selectin binding. Little information is available on the intra- and inter-species evolution of PSGL-1 primary structure. In addition, the evolutionary conservation of selectin binding site on PSGL-1 has not been previously examined in detail. Therefore, we performed multiple sequence alignment of PSGL-1 amino acid sequences of 14 mammals (human, chimpanzee, rhesus monkey, bovine, pig, rat, tree-shrew, bushbaby, mouse, bat, horse, cat, sheep and dog) and examined mammalian PSGL-1 interactions with human selectins.ResultsA signal peptide was predicted in each sequence and a propeptide cleavage site was found in 9/14 species. PSGL-1 N-terminus is poorly conserved. However, each species exhibits at least one tyrosine sulfation site and, except in horse and dog, a T [D/E]PP [D/E] motif associated to the core-2 O-glycosylation of a N-terminal threonine. A mucin-like domain of 250–280 amino acids long was disclosed in all studied species. It lies between the conserved N-terminal O-glycosylated threonine (Thr-57 in human) and the transmembrane domain, and contains a central region exhibiting a variable number of decameric repeats (DR). Interspecies and intraspecies polymorphisms were observed. Transmembrane and cytoplasmic domain sequences are well conserved. The moesin binding residues that serve as adaptor between PSGL-1 and Syk, and are involved in regulating PSGL-1-dependent rolling on P-selectin are perfectly conserved in all analyzed mammalian sequences. Despite a poor conservation of PSGL-1 N-terminal sequence, CHO cells co-expressing human glycosyltransferases and human, bovine, pig or rat PSGL-1 efficiently rolled on human L- or P-selectin. By contrast, pig or rat neutrophils were much less efficiently recruited than human or bovine neutrophils on human selectins. Horse PSGL-1, glycosylated by human or equine glycosyltransferases, did not interact with P-selectin. In all five species, tyrosine sulfation of PSGL-1 was required for selectin binding.ConclusionThese observations show that PSGL-1 amino acid sequence of the transmembrane and cytoplasmic domains are well conserved and that, despite a poor conservation of PSGL-1 N-terminus, L- and P-selectin binding sites are evolutionary conserved. Functional assays reveal a critical role for post-translational modifications in regulating mammalian PSGL-1 interactions with selectins.

A Template‐Assembled Synthetic Protein Surface Mimetic of the von Willebrand Factor A1 domain Inhibits Botrocetin‐Induced Platelet Aggregation

Platelet adhesion, the initial step of platelet activation, is mediated by the interaction of von Willebrand factor (VWF) with its platelet receptor, the GPIb–IX complex. The binding of VWF to GPIb–IX is induced either by increased shear stress or by exogenous modulators, such as botrocetin. At a molecular level, this interaction takes place between the A1 domain of VWF and the GPIbα chain of the GPIb–IX complex. We report here the design and functional characteristics of a VWF template‐assembled synthetic protein (TASP), a chimeric four‐helix‐bundle TASP scaffold mimicking the surface of the A1 domain. Twelve residues located on helices α3 and α4 in the native A1 domain were grafted onto a surface formed by two neighboring helices of the TASP. VWF TASP was found to inhibit specifically botrocetin‐induced platelet aggregation and to bind both botrocetin and GPIbα. However, in contrast to the native A1 domain, VWF TASP did not bind simultaneously to both ligands. Modeling studies revealed that the relative orientation of the α helices in VWF TASP led to a clash of bound botrocetin and GPIbα. These results demonstrate that a chimeric four‐helix‐bundle TASP as a scaffold offers a suitable surface for presenting crucial residues of the VWF A1 domain; the potential of the TASP approach for de novo protein design and mimicry is thereby illustrated.

Acute myeloid leukemia in the elderly: Results of an individualized approach in two centres

We retrospectively assessed seventy-four consecutive patients with AML over 65 years of age (median 71: range 65-88) treated with an individualized approach in two specialized cancer centers. Patients were managed according to their performance status (PS) and associated discases in both institutions. The proportion of patients with poor PS (3-4) was higher in center I (37%) than in center 2 (10%) and in center I palliative treatment was given more frequently (16/32 patients) than in center 2 (7/42 patients). Fifty-one patients received intensive combination chemotherapy including an anthracycline and ara-C or VP16 (2 patients) and 36 patients received a second intensive course as reinduction or as consolidation treatment after complete remission. Patients not eligible for myelosuppressive chemotherapy were treated with palliative measures (23 patients). With intensive chemotherapy, complete remission (CR) was achieved in 29 of 51 patients (57%). after first (20 patients) or second course (9 patients) and the rate of deaths during induction was 14% (7 patients). The CR rate was lower for patients with performance status ≥ 2 (48%) as compared to patients with performance status ≤ 1 (78%) and for patients with secondary AML (46%) as compared to patients with de novo AML (60%). Median survival was 9.1 months for patients receiving intensive treatment and 1.2 months for patients receiving palliative treatment (P=0.001). In the Cox model for overall survival. treatment with curative intent was associated with longer survival (hazard ratio: 0.33, 95% confidence interval: 0.18-0.58) and poor performance status (PS ≥ 2) was associated with reduced survival (hazard ratio: 3.29. 95%. confidence interval: 1.72-6.17). Overall 2-years and 5-years survival were 20% and 11% for the patients treated intensively. From this study it appears that an individualized approach of treatment with intensive chemotherapy for selected patients offers a substantial CR rate and an improvement in survival. This analysis also suggests that differences in outcome between single institutions can be explained mainly by referral and selection biases

P-selectin Glycoprotein Ligand-1 Decameric Repeats Regulate Selectin-dependent Rolling under Flow Conditions

P-selectin glycoprotein ligand-1 (PSGL-1) interacts with selectins to support leukocyte rolling along vascular wall. L- and P-selectin bind to N-terminal tyrosine sulfate residues and to core-2 O-glycans attached to Thr-57, whereas tyrosine sulfation is not required for E-selectin binding. PSGL-1 extracellular domain contains decameric repeats, which extend L- and P-selectin binding sites far above the plasma membrane. We hypothesized that decamers may play a role in regulating PSGL-1 interactions with selectins. Chinese hamster ovary cells expressing wild-type PSGL-1 or PSGL-1 molecules exhibiting deletion or substitution of decamers with the tandem repeats of platelet glycoprotein Ibα were compared in their ability to roll on selectins and to bind soluble L- or P-selectin. Deletion of decamers abrogated soluble L-selectin binding and cell rolling on L-selectin, whereas their substitution partially reversed these diminutions. P-selectin-dependent interactions with PSGL-1 were less affected by decamer deletion. Videomicroscopy analysis showed that decamers are required to stabilize L-selectin-dependent rolling. Importantly, adhesion assays performed on recombinant decamers demonstrated that they directly bind to E-selectin and promote slow rolling. Our results indicate that the role of decamers is to extend PSGL-1 N terminus far above the cell surface to support and stabilize leukocyte rolling on L- or P-selectin. In addition, they function as a cell adhesion receptor, which supports ∼80% of E-selectin-dependent rolling.

Treatment of 5q-syndrome with lenalidomide in an HIV-positive patient under cART

Dear Editor, Lenalidomide has emerged as standard treatment for 5qsyndrome [1]. In normal cell cultures, lenalidomide enhances T cell proliferation [2], and in cells isolated from HIV-positive (HIV+) and CMV+ patients, lenalidomide enhances CD8+ cytotoxic T cell activity against viral antigens [3]. There are no in vivo data on lenalidomide in HIV+ patients. We are reporting on a 52-year-old HIV+ patient with an undetectable viral load for the past 10 years on combined antiretroviral therapy (cART) who developed 5q-syndrome and was successfully treated with lenalidomide. In 1989, he was diagnosed with HIV and had been on cART since 1996 after monoand bi-therapies. He had suffered and recovered from multiple complications (CMV-retinitis, Kaposi sarcoma, cryptococcal meningitis, oesophageal candidiasis, disseminated Mycobacterium genavense infection and anal squamous cell carcinoma (T1N0M0)). In February 2007, persistent pancytopenia and dependency on red blood cell transfusions for a month motivated a bone marrow (BM) biopsy revealing a 5q-syndrome (karyotype: 46,XY,del(5) (q13-14q33)[7]/46,XY[3]). In August 2007 lenalidomide was started (10 mg for 21 days every 28 days) according to recommendations on the use of lenalidomide in MDS without dose reduction at any time [4]. He became transfusion independent 18 days after treatment start. After two cycles, complete normalisation of peripheral blood counts was achieved (Fig. 1), and phlebotomy therapy was initiated for iron overload. After five cycles, BM examination revealed complete cytological and partial cytogenetic remission (5q deletion in eight of 50 metaphases). HIV viral load remained undetectable before lenalidomide and 1, 2, 3, 4, 8, 12 and 24 weeks after the start of lenalidomide. CD4+ cells had reached a steady state between 137 and 206 cells/mm before lenalidomide. They rose to 235 cells/mm 2 months after starting lenalidomide and remained stable between 230 and 240 cells/mm. CD8+ cells were between 314 and 393 cells/mm before and reached stable levels over 500 cells/mm 6 weeks after treatment began (527–1,195 cells/mm). Lymphocyte counts rose from 0.9 to 1×10/l to stable levels between 1.2 and 2.5×10/l 3 months after treatment was initiated (Figs. 1 and 2). Before lenalidomide treatment, plasma concentrations of atazanavir (ATV) and lopinavir (LPV) were 1,767 and 13,903 ng/ml 5 h after last drug intake. On days 23 and 28 of cycle2 of lenalidomide treatment, plasma levels 11.5 h after last drug intake were 2,728 and 2,180 ng/ml for ATV and 7,728 and 5,791 ng/ml for LPV, respectively. At day20 S. Blum (*) : J.-F. Lambert :M. Schapira Service of Haematology, Centre Hospitalier Universitaire Vaudois, Rue du Bugnon 46, 1011 Lausanne, Switzerland e-mail: sabine_blum@bluewin.ch

Design and synthesis of a chimeric TASP molecule as potential inhibitor in cell adhesion processes

The construction of protein-like folding motifs as structurally stable scaffolds for the introduction of function represents a major goal in protein design. The use of topol. templates allows the bypass of the well-known folding problem of linear polypeptides and offers a way to mimic native packing topologies by the template directed self-assembly of helical and/or b-sheeted peptide blocks. In conceptually sepg. structure from function, a chimeric 4-helix bundle TASP (Template Assembled Synthetic Protein) derived from the ROP protein and the cell adhesion glycoprotein E-selectin has been designed and synthesized, aimed at inhibiting an early stage in cell adhesion processes, in particular leukocyte adhesion. [on SciFinder (R)]