Kerstin Danker

Biochemical engineering of the acyl side chain of sialic acids stimulates integrin-dependent adhesion of HL60 cells to fibronectin

Abstract. Sialylation of glycoproteins and glycolipids plays an important role during development, regeneration and pathogenesis of several diseases. The physiological precursor of all sialic acids is N-acetyl-D-mannosamine. The N-acyl side chain of sialic acid can be modified by exposure of cells to synthetic N-acyl-modified D-mannosamines. In a new experimental approach cells were cultivated in the presence of N-propanoyl-D-mannosamine. This unnatural precursor of sialic acid is taken up by cells and efficiently metabolized to the respective N-acyl-modified neuraminic acid in vitro and in vivo. Here we report on the biological consequences of the incorporation of the unnatural N-propanoylneuraminic acid into glycoconjugates of HL60 cells. Biochemical engineering of the acyl side chain of neuraminic acids activates β1-integrins (VLA4 or VLA5), resulting in an increased adhesion of HL60 cells to fibronectin.

Protein kinase C phosphorylates and regulates UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase

UDP‐N‐acetylglucosamine‐2‐epimerase/N‐acetylmannosamine kinase (UDP‐GlcNAc 2‐epimerase) is the key enzyme in the de novo synthesis pathway of neuraminic acid, which is widely expressed as a terminal carbohydrate residue on glycoconjugates. UDP‐GlcNAc 2‐epimerase is a bifunctional enzyme and catalyzes the first two steps of neuraminic acid synthesis in the cytosol, the conversion of UDP‐N‐acetylglucosamine to ManAc and the phosphorylation to ManAc‐6‐phosphate. So far, regulation of this essential enzyme by posttranslational modification has not been shown. Since UDP‐N‐acetylglucosamine is a cytosolic protein containing eight conserved motifs for protein kinase C (PKC), we investigated whether its enzymatic activity might be regulated by phosphorylation by PKC. We showed that UDP‐GlcNAc 2‐epimerase interacts with several isoforms of PKC in mouse liver and is phosphorylated in vivo. Furthermore, PKC phosphorylates UDP‐GlcNAc 2‐epimerase and this phosphorylation results in an upregulation of the UDP‐GlcNAc 2‐epimerase enzyme activity.

alpha1 Integrin cytoplasmic domain is involved in focal adhesion formation via association with intracellular proteins.

Integrins are heterodimeric adhesion receptors consisting of alpha- and beta-subunits capable of binding extracellular matrix molecules as well as other adhesion receptors on neighbouring cells. These interactions induce various signal transduction pathways in many cell types, leading to cytoskeletal reorganization, phosphorylation and induction of gene expression. Integrin ligation leads to cytoplasmic protein-protein interactions requiring both integrin cytoplasmic domains, and these domains are initiation points for focal adhesion formation and subsequent signal transduction cascades. In previous studies we have shown that the very short cytoplasmic alpha1 tail is required for post-ligand events, such as cell spreading as well as actin stress-fibre formation. In the present paper we report that cells lacking the cytoplasmic domain of the alpha1 integrin subunit are unable to form proper focal adhesions and that phosphorylation on tyrosine residues of focal adhesion components is reduced on alpha1beta1-specific substrates. The alpha1 cytoplasmic sequence is a specific recognition site for focal adhesion components like paxillin, talin, alpha-actinin and pp125FAK. It seems to account for alpha1-specific signalling, since when peptides that mimic the cytoplasmic domain of alpha1 are transferred into cells, they influence alpha1beta1-specific adhesion, presumably by competing for binding partners. For alpha1 integrin/protein binding, the conserved Lys-Ile-Gly-Phe-Phe-Lys-Arg motif and, in particular, the two lysine residues, are important.

V+-Fibronectin expression and localization prior to gastrulation in Xenopus laevis embryos

The V-region represents one of three alternatively spliced segments in Xenopus fibronectin. Here, we identify this V-region as binding epitope of the monoclonal antibody (MAb 6D9) that we generated against Xenopus plasma fibronectin. By the use of this antibody we obtained new results that change the present view of the fibronectin expression pattern before gastrulation: (1) the V(+)-fibronectin is the major isoform expressed during early development since only a single fibronectin band is found in Western blots up to tadpole stages. (2) In contrast to previously published data we demonstrate that fibronectin expression is induced by progesterone during oocyte maturation. (3) During cleavage stages the protein is stored in the cytoplasm where it is predominantly associated with plasma membranes. Immunoelectronmicroscopy reveals that V(+)-fibronectin is present at the surface of animal pole blastomeres and secreted into intercellular spaces. This extracellular localization of fibronectin is predominantly observed in the marginal zone, surrounding single cells of the outer cell layer baso-laterally. In the vegetal hemisphere V(+)-fibronectin is restricted to the cytoplasm and accumulated at plasma membranes. With the onset of gastrulation the intracellular and membrane associated fibronectin disappears and fibronectin becomes detectable at the blastocoel roof. Since reaggregation of dissociated blastula cells was not blocked by addition of GRGDS peptide or antibodies against fibronectin, we assume that the early expression and secretion of fibronectin serves as store to allow a rapid matrix assembly with onset of mesodermal cell migration.

Carcinoembryonic antigen-related cell-cell adhesion molecule C-CAM is greatly increased in serum and urine of rats with liver diseases

C‐CAM (rat cell CAM/human CD66a) is ubiquitous and multifunctional. It is involved in intercellular adhesion, signal transduction and cell growth inhibition. Structurally, it is related to the carcinoembryonic antigen. In the present study serum, bile and urine of rats with liver diseases were analyzed for the presence of cell CAM. After bile duct ligation and during galactosamine (GalN) hepatitis we found that large amounts of liver membrane‐bound C‐CAM are secreted or shed into blood. The serum level of another liver membrane‐bound protein, LI‐cadherin, is not increased. It was shown that C‐CAM is also present in bile fluid, and for the first time that C‐CAM is present in the urine of rats with liver diseases. A particularly high concentration was measured in the urine of rats suffering from GalN hepatitis.

Phospholipase Cgamma binds alpha1beta1 integrin and modulates alpha1beta1 integrin-specific adhesion.

Integrin adhesion receptors have been implicated in bidirectional signal transduction. The dynamic regulation of integrin affinity and avidity as well as post-ligand effects involved in outside-in signaling depends on the interaction of integrins with cytoskeletal and signaling proteins. In this study, we attempted to identify cytoplasmic binding partners of α1β1 integrin. We were able to show that cell adhesion to α1β1-specific substrates results in the association of phospholipase Cγ (PLCγ) with the α1β1 integrin independent of PLCγ tyrosine phosphorylation. Using peptide-binding assays, the membrane proximal sequences within the α1β1integrin subunits were identified as binding sites for PLCγ. In particular, the conserved sequence of β1 subunit binds the enzyme very efficiently. Because purified PLCγ also binds the integrin peptides, binding seems to be direct. Inhibition of PLC byU73122 leads to reduced cell adhesion on α1β1-specific substrates. Cells lacking the conserved domain of the α1 subunit fail to respond to the PLC inhibition, indicating that this domain is necessary for PLC-dependent adhesion modulation of α1β1 integrin.

Phospholipase Cγ Binds α1β1Integrin and Modulates α1β1 Integrin-specific Adhesion

Integrin adhesion receptors have been implicated in bidirectional signal transduction. The dynamic regulation of integrin affinity and avidity as well as post-ligand effects involved in outside-in signaling depends on the interaction of integrins with cytoskeletal and signaling proteins. In this study, we attempted to identify cytoplasmic binding partners of α1β1 integrin. We were able to show that cell adhesion to α1β1-specific substrates results in the association of phospholipase Cγ (PLCγ) with the α1β1 integrin independent of PLCγ tyrosine phosphorylation. Using peptide-binding assays, the membrane proximal sequences within the α1β1integrin subunits were identified as binding sites for PLCγ. In particular, the conserved sequence of β1 subunit binds the enzyme very efficiently. Because purified PLCγ also binds the integrin peptides, binding seems to be direct. Inhibition of PLC byU73122 leads to reduced cell adhesion on α1β1-specific substrates. Cells lacking the conserved domain of the α1 subunit fail to respond to the PLC inhibition, indicating that this domain is necessary for PLC-dependent adhesion modulation of α1β1 integrin.