Geertruida M. Veldman

Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis

Many mammalian viruses have acquired genes from their hosts during their evolution. The rationale for these acquisitions is usually quite clear: the captured genes are subverted to provide a selective advantage to the virus. Here we describe the opposite situation, where a viral gene has been sequestered to serve an important function in the physiology of a mammalian host. This gene, encoding a protein that we have called syncytin, is the envelope gene of a recently identified human endogenous defective retrovirus, HERV-W. We find that the major sites of syncytin expression are placental syncytiotrophoblasts, multinucleated cells that originate from fetal trophoblasts. We show that expression of recombinant syncytin in a wide variety of cell types induces the formation of giant syncytia, and that fusion of a human trophoblastic cell line expressing endogenous syncytin can be inhibited by an anti-syncytin antiserum. Our data indicate that syncytin may mediate placental cytotrophoblast fusion in vivo, and thus may be important in human placental morphogenesis.

Expression cloning of a functional glycoprotein ligand for P-selectin

The initial adhesive interactions between circulating leukocytes and endothelia are mediated, in part, by P-selectin. We now report the expression cloning of a functional ligand for P-selectin from an HL-60 cDNA library. The predicted amino acid sequence reveals a novel mucin-like transmembrane protein. Significant binding of transfected COS cells to P-selectin requires coexpression of both the protein ligand and a fucosyltransferase. This binding is calcium dependent and can be inhibited by a neutralizing monoclonal antibody to P-selectin. Cotransfected COS cells express the ligand as a homodimer of 220 kd. A soluble ligand construct, when coexpressed with fucosyltransferase in COS cells, also mediates P-selectin binding and is immunocrossreactive with the major HL-60 glycoprotein that specifically binds P-selectin.

Inhibition of myostatin in adult mice increases skeletal muscle mass and strength

A human therapeutic that specifically modulates skeletal muscle growth would potentially provide a benefit for a variety of conditions including sarcopenia, cachexia, and muscular dystrophy. Myostatin, a member of the TGF-beta family of growth factors, is a known negative regulator of muscle mass, as mice lacking the myostatin gene have increased muscle mass. Thus, an inhibitor of myostatin may be useful therapeutically as an anabolic agent for muscle. However, since myostatin is expressed in both developing and adult muscles, it is not clear whether it regulates muscle mass during development or in adults. In order to test the hypothesis that myostatin regulates muscle mass in adults, we generated an inhibitory antibody to myostatin and administered it to adult mice. Here we show that mice treated pharmacologically with an antibody to myostatin have increased skeletal muscle mass and increased grip strength. These data show for the first time that myostatin acts postnatally as a negative regulator of skeletal muscle growth and suggest that myostatin inhibitors could provide a therapeutic benefit in diseases for which muscle mass is limiting.

Program Death-1 Engagement Upon TCR Activation Has Distinct Effects on Costimulation and Cytokine-Driven Proliferation: Attenuation of ICOS, IL-4, and IL-21, But Not CD28, IL-7, and IL-15 Responses

The program death 1 (PD-1) receptor and its ligands, PD-1 ligand (PD-L)1 and PD-L2, define a novel regulatory pathway with potential inhibitory effects on T, B, and monocyte responses. In the present study, we show that human CD4+ T cells express PD-1, PD-L1, and PD-L2 upon activation, and Abs to the receptor can be agonists or antagonists of the pathway. Under optimal conditions of stimulation, ICOS but not CD28 costimulation can be prevented by PD-1 engagement. IL-2 levels induced by costimulation are critical in determining the outcome of the PD-1 engagement. Thus, low to marginal IL-2 levels produced upon ICOS costimulation account for the greater sensitivity of this pathway to PD-1-mediated inhibition. Interestingly, exogenous IL-2, IL-7, and IL-15 but not IL-4 and IL-21 can rescue PD-1 inhibition, suggesting that among these cytokines only those that activate STAT5 can rescue PD-1 inhibition. As STAT5 has been implicated in the maintenance of IL-2Rα expression, these results suggest that IL-7 and IL-15 restore proliferation under conditions of PD-1 engagement by enhancing high-affinity IL-2R expression and hence, IL-2 responsiveness.

P-selectin Glycoprotein Ligand-1 Is the Major Counter-receptor for P-selectin on Stimulated T Cells and Is Widely Distributed in Non-functional Form on Many Lymphocytic Cells

P-selectin glycoprotein ligand-1 (PSGL-1) is the high affinity counter-receptor for P-selectin on myeloid cells (Sako, D., Chang, X. J., Barone, K. M., Vachino, G., White, H. M., Shaw, G., Veldman, G. M., Bean, K. M., Ahern, T. J., Furie, B., Cumming, D. A., and Larsen, G. R.(1993) Cell 75, 1179-1186). Here we demonstrate that PSGL-1 is also widely distributed on T- and B-lymphocytic tumor cell lines, resting peripheral blood T and B cells, and on stimulated peripheral blood T cell and intestinal intraepithelial lymphocyte (IEL) lines. However, the majority of PSGL-1-positive resting peripheral blood lymphocytic cells and lymphoid tumor cell lines do not display significant P-selectin binding. In contrast, in vitro stimulated peripheral blood T cell and IEL lines avidly bind P-selectin, and PSGL-1 is the sole high affinity counter-receptor mediating this binding. During the course of in vitro stimulation, cell surface expression levels of PSGL-1 do not change as P-selectin binding increases. Rather, the activities of two glycosyltransferases reportedly involved in the production of functional PSGL-1 in myeloid cells are substantially higher in the stimulated T-lymphocytic lines than in resting T lymphocytes, consistent with the hypothesis that activation-dependent post-translational events contribute to the expression of functional PSGL-1 on lymphocytes.

A Novel Cobra Venom Metalloproteinase, Mocarhagin, Cleaves a 10-Amino Acid Peptide from the Mature N Terminus of P-selectin Glycoprotein Ligand Receptor, PSGL-1, and Abolishes P-selectin Binding

Initial rolling of circulating neutrophils on a blood vessel wall prior to adhesion and transmigration to damaged tissue is dependent upon P-selectin expressed on endothelial cells and its specific neutrophil receptor, the P-selectin glycoprotein ligand-1 (PSGL-1). Pretreatment of neutrophils, HL60 cells, or a recombinant fucosylated soluble form of PSGL-1 (sPSGL-1.T7) with the cobra venom metalloproteinase, mocarhagin, completely abolished binding to purified P-selectin in a time-dependent and EDTA- and diisopropyl fluorophosphate-inhibitable manner consistent with mocarhagin selectively cleaving PSGL-1. A polyclonal antibody against the N-terminal peptide Gln-1-Glu-15 of mature PSGL-1 immunoprecipitated sPSGL-1.T7 but not sPSGL-1.T7 treated with mocarhagin, indicating that the mocarhagin cleavage site was near the N terminus. A single mocarhagin cleavage site between Tyr-10 and Asp-11 of mature PSGL-1 was determined by N-terminal sequencing of mocarhagin fragments of sPSGL-1.T7 and is within a highly negatively charged amino acid sequence 1-QATEYEYLDYDFLPETEPPE, containing three tyrosine residues that are consensus sulfation sites. Consistent with a functional role of this region of PSGL-1 in binding P-selectin, an affinity-purified polyclonal antibody against residues Gln-1-Glu-15 of PSGL-1 strongly inhibited P-selectin binding to neutrophils, whereas an antibody against residues Asp-9-Arg-23 was noninhibitory. These combined data strongly suggest that the N-terminal anionic/sulfated tyrosine motif of PSGL-1 as well as downstream sialylated carbohydrate is essential for binding of P-selectin by neutrophils.