Thrombomodulin (TM) on the endothelial cell surface is a cofactor protein that modulates the substrate specificity of thrombin and promotes protein C activation. Previous studies have shown that rabbit and recombinant human soluble TMs contain a chondroitin sulfate moiety attached to a Ser/Thr-rich domain. In the case of recombinant human soluble TM produced in Chinese hamster ovary (CHO) cells, this glycosaminoglycan (GAG) is composed of chondroitin 4-sulfate as a major component. The modification is not required for the cofactor activity, but contributes to ability of TM to inhibit procoagulant activities of thrombin (fibrionogen clotting, platelet activation and factor V activation) and also affects inactivation of thrombin by antithrombin III (AT III). Since TM modified with non-sulfated GAG (chondroitin) has functions similar to those of TM lacking in GAG modification, it is obvious that the sulfation of the attached GAG is required for these activities. Consistent with in vitro studies, recombinant human soluble TM modified with chondroitin 4-sulfate (rsTMβ) exhibited more potent anticoagulant activity in a rat thrombosis (tissue factor-induced disseminated intravascular coagulation; DIC) model than the unmodified form. rsTMβ thus appears to be an effective antithrombotic agent for inhibiting DIC. In the cellular expression system of introduced TM gene, the host cell has ability to determine the location of sulfate groups on the amino sugars constituting GAG chain. Native TM on cultured human umbilical vein endothelial cells as well as recombinant human soluble TM contains GAG-modified form.
CD22 is a B lymphocyte-specific adhesion molecule expressed on mature B cells. Recent work has shown that although CD22 has the structure of an immunoglobulin superfamily member, its functional properties are those of a sialic acid binding lectin. The uniqueness of CD22 is underscored by the observation that it recognizes sialic acid in a specific linkage only and that sialylation of CD22 itself by α2, 6 sialyltransferase can abrogate its function as an adhesion molecule. The discovery that the leukocyte-specific phosphotyrosine phosphatase, CD45, is among sialoglycoproteins recognized by CD22, adds to the potential interest of CD22 as a molecule that may participate in the regulation of lymphocyte activation. This review will summarize our current understanding of the CD22 molecule and its putative functions.
Hyaluronan (HA) is an evolutionarily conserved, large and negatively charged glycosaminoglycan that has been implicated in the locomotion of cells during morphogenesis, wound repair, and disease states such as tumorigenesis. Recently, we demonstrated that HA is produced in elevated levels in ras-transformed cells and is required for their motility. Furthermore, TGF-β1 which stimulates locomotion of these cells requires HA for this effect. We have shown that RHAMM, a novel glycoprotein that was recently cloned from fibroblasts, mediates the locomotion of ras-transformed fibroblasts. Thus, blocking antibodies inhibit ras-transformed locomotion and conversely, cells transfected with a genomic RHAMM clone, that overexpress cell surface RHAMM, exhibit a two fold increased in cell locomotion and also display a loss of contact inhibition of locomotion, as demonstrated by a nuclear overlap ratio, and of growth as demonstrated by overgrowth at culture confluence. The mechanism(s) by which HA might regulate cell locomotion and contact inhibition are unknown. HA promotes transient increases in protein tyrosine phosphorylation, a phenomenon that is required for cell locomotion to occur. Concomitant with an increase in protein tyrosine phosphorylation is an increase in lamellae extension and the transient appearance of small point focal contacts. These structures show increased protein tyrosine content and contain focal adhesion kinase (FAK). Focal contacts disappear 10-15min after HA stimulation as cells begin to displace, suggesting that a key role of HA in stimulating locomotion is the regulation of focal contact turnover. Our results provide evidence for an important role of HA and of one of its HA receptors, RHAMM, in the contact and motility behavior of tumor cells and predict that these molecules play a critical role in tumor progression.
A number of recent findings concerning the metabolism of sphingolipids (SL) and the role of SLs and glycosphingolipids in various aspects of cell function are discussed in this mini-review. Particular attention is paid to three areas, namely i) the intracellular sites and topology of SL synthesis, ii) the transport of SLs by vesicular and non-vesicular mechanisms through the secretory pathway, and iii) the physiological consequences of inhibiting SL synthesis and of depleting SL levels. Experimental strategies are suggested that may yield useful information to advance further our understanding of the roles that sphingolipids may play in cell function.