Syndecan interacting proteins and the use thereof

Details Syndecan interacting proteins and the use thereof Syndecan interacting proteins and the use thereof

1999-10-25

2002-04-09

Carlson, Karen Cochrane (Department: 1653)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C530S300000, C530S350000

Reexamination Certificate

active

06368811

ABSTRACT:

TECHNICAL FIELD
The present invention relates to syndecan interacting protein(s) and the use thereof.
BACKGROUND
The invention relates to the identification of a protein that binds to the cytoplasmic domain of the syndecan. This protein, now called syntenin, contains a tandem repeat of PDZ-domains that reacts with the FYA C-terminal amino acid sequence of the syndecans (Trends Biochem.Sci., 20, p. 350, 1995: Origin of PDZ {DHR, GLGF}, by M. B. Kennedy). Endogenous syntenin appears to be localized to the cytoskeleton.
DISCLOSURE OF THE INVENTION
GFP-syntenin fusion-proteins decorate the plasma membrane and intracellular vesicles, where they co-localize and -segregate with syndecan cytoplasmic domains. Syntenin, therefore, is an unexpected candidate for connecting the cytoskeleton to heparan sulfate-assisted signal transduction pathways.
The syndecan cytoplasmatic link (sycl) protein or syndecan interacting protein is referred to by the name “syntenin”, meaning “putting tension on the syndecans.”
Heparan sulfate proteoglycans (HSPG) are proteins which are mostly associated with the cell membrane. The most characterizing feature of these proteins is that they are substituted with different heparan sulfate sugar derivatives which strongly determine the function of said protein. Basically there are two classes of membrane heparan sulfate proteoglycans. First the best characterized membrane proteoglycans are the syndecans, which have a membrane spanning core protein. The second class is the glypican family which are located at the cell surface and anchored in the cell membrane through GPI (glycosyl phosphatidyl inositol).
The existence of two distinct highly conserved multigene families of cell surface proteoglycans, the syndecan and glypican families respectively, suggests two distinctive cellular and/or subcellular pathways wherein these proteins will function. Ramifications within these pathways that are specified by the variations on a basic structural theme are realized by the various members of these families. The aforementioned HSPGs are involved in several physiological processes and play a definite a role in the transmission of signals from outside a cell into the cell itself. Their their activities are characterized by the specific binding of heparan sulfate molecules to proteinase inhibitors, cell adhesion molecules or growth factors.
If the structure of HSPG is disturbed, abnormal cell growth and abnormal morphogenesis occur. Therefore it is of utmost importance to know and understand the structure-function relation of the proteoglycans and the way they transmit outside signals through the cell membrane into the cell, the so-called signal transduction cascade.
As mentioned above syndecans are transmembrane proteoglycans that place structurally heterogeneous heparan sulfate chains at the cell surface and a highly conserved polypeptide in the cytoplasm. Their versatile heparan sulfate moieties support various processes of molecular recognition, signaling and trafficking.
The cell surface heparan sulfate proteoglycans are at the cross-section of several different pathways. Their heparan sulfate moieties bind various differentiation-, growth-, and scatter factors, facilitate the occupation and activation of the corresponding signal-transducing receptors, and are involved in the internalization and clearance of the signaling complexes from the cell surface. They also assist receptors that are involved in cell—cell and cell-matrix adhesion, and assist scavenging receptors that are involved in the endocytosis and transcytosis of lipoproteins and lipases. They also bind and activate serine proteinase inhibitors and accelerate the reactions of these inhibitors with their targets. Proteolysis, lipolysis, mesoderm-induction, gastrulation, angiogenesis, neuritogenesis all appear to be regulated by or to depend on heparan sulfate, because this glycosaminoglycan is needed for the allosteric activation, approximation and compartmentalization of the reactants that are engaged in these processes.
In most cells syndecans represent the major source of cell surface heparan sulfate. The four known syndecans are small type I membrane proteins, with similar and simple domain organizations: a single ectodomain, membrane-span, and cytoplasmic domain. Except for the presence of three or four consensus sites for heparan sulfate attachment, near the amino-termini of the proteins, and a dibasic, presumably protease-sensitive site at the junctions with the membrane spanning segments, the ectodomains of the different syndecans have little in common. The structures of these ectodomains have also not been evolutionary conserved, except for these shared structural elements. The membrane-spanning and the small cytoplasmic domains of the syndecans, in contrast, show extensive structural similarity (60% sequence identity) and have been highly conserved during evolution. All four vertebrate syndecans and the single Drosophila syndecan share the amino acid sequence RM(K/R)KKDEGSY—depicted in one-letter code— in the membrane-proximal segments of their cytoplasmic domains, and the amino acid sequence EFYA —depicted in one letter code— at their C-termini. This suggests that the extracellular heparan sulfate moieties, the cytoplasmic protein moieties, and the contiguity of these moieties are essential for syndecan function. The syndecans may provide for a transmembrane link between extracellular heparin-steered processes and intracellular structural or regulatory proteins, and mediate outside-in or inside-out effects on signaling or effector systems.
BEST MODE OF THE INVENTION
In order to understand above mechanism/cascade a search for Syndecan cytoplasmic links (sycls) was initiated using the cytoplasmic domains of four different syndecans as baits in a yeast two-hybrid screening assay. The insert of one apparent truly positive clone (yielding HIS+ and LacZ+ phenotypes in combination with all four syndecan/Gal4 DNA-binding domain fusion constructs, but not with Gal4 DNA-binding domain alone or with p53 fused to the Gal4 DNA-binding domain) was sequenced, and used as a starting point to obtain a cDNA coding for the corresponding full length sycl protein.
The present invention concerns syndecan interacting protein(s) obtainable by a two-hybrid screening assay whereby as bait a cytoplasmic domain comprising the amino acid sequence FYA as C-terminal sequence as occurring in syndecan and as prey a cDNA library is used. For the purpose of the invention the cDNA library can be any suitable cDNA library, but preferably be a mammal, more preferably a human and most preferably a human liver cDNA library. The fall length sycl thus obtained consists of 298 aminoacids, and can be divided in three or four parts. The first amino-terminal region (aa 1-109) shows no striking homology to any known structural motif. It is relatively rich in proline and contains five tyrosines, while the remainder of the protein is free of tyrosine. Based on sequence alignments, the second (aa 101-193) and third (aa 194-274) regions of sycl appear to correspond to a tandem repeat of two PDZ domains. The sequence coding for the putative second PDZ domain is extended by 24 amino acids (aa 275-298), which may still be part of the second PDZ domain or compose a fourth separate C-terminal domain. PDZ domains have recently been recognized as one of the conserved modular structures that support protein—protein interactions and networking. PDZ domains mediate protein—protein interactions by binding to the carboxy-terminal ends of target proteins, and often occur in association with other functional modules, such as SH3 domains, protein tyrosine phosphatase domains, domains related to guanylate kinase (GUK), to band 4.1 protein, leucine zipper motifs, and additional PDZ domains. PDZ domains have now been discovered in a variety of proteins, and shown to bind to membrane channels, receptors (e.g. wingless and Notch), tumor supressor proteins (APC), GAPs and GEFs. These interactions appear to be involved in the formation of multimeric pro

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