Altered localization and cytoplasmic domain-binding properties of tyrosine-phosphorylated beta 1 integrin.

We describe a novel approach to study tyrosine-phosphorylated (PY) integrins in cells transformed by virally encoded tyrosine kinases. We have synthesized a peptide (PY beta 1 peptide) that represents a portion of the cytoplasmic domain of the beta 1 integrin subunit and is phosphorylated on the tyrosine residue known to be the target of oncogenic tyrosine kinases. Antibodies prepared against the PY beta 1 peptide, after removal of cross-reacting antibodies by absorption and affinity purification, recognized the PY beta 1 peptide and the tyrosine-phosphorylated form of the intact beta 1 subunit, but did not bind the nonphosphorylated beta 1 peptide, the nonphosphorylated beta 1 subunit or other unrelated tyrosine-phosphorylated proteins. The anti-PY beta 1 antibodies labeled the podosomes of Rous sarcoma virus-transformed fibroblasts, but did not detectably stain nontransformed fibroblasts. The localization of the tyrosine phosphorylated beta 1 subunits appeared distinct from that of the beta 1 subunit. Adhesion plaques were stained by the anti-beta 1 subunit antibodies in Rous sarcoma virus-transformed fibroblasts plated on fibronectin, whereas neither podosomes nor adhesion plaques were labeled on vitronectin or on uncoated plates. Anti-phosphotyrosine antibodies labeled podosomes, adhesion plaques and cell-cell boundaries regardless of the substratum. One of the SH2 domains of the p85 subunit of phosphatidylinositol-3-kinase bound to the PY beta 1 peptide, but not to the non-phosphorylated beta 1 cytoplasmic peptide. Other SH2 domains did not bind to the PY beta 1 peptide. These results show that the phosphorylated form of the beta 1 integrin subunit is detected in a different subcellular localization than the nonphosphorylated form and suggest that the phosphorylation on tyrosine of the beta 1 subunit cytoplasmic domain may affect cellular signaling pathways.

Biochemical properties of tissue polypeptide antigen.

Tissue polypeptide antigen (TPA) is a complex protein which has been originally identified in extracts of pooled tumors using horse antisera raised against the insolubles of human tumor cells. The antigen is now routinely detected and measured by a previously described hemagglutination inhibition assay. It has been shown by this method that the concentration of the antigen is higher in tumor tissues and in sera of cancer patients as compared to normal tissues or normal sera, respectively. In aqueous solutions, pH 2-12, TPA has a tendency to form high molecular weight aggregates. However they can be dissociated in sodium dodecyl sulfate into subunits, each appearing as a single chain peptide: B1 (Mr 4.3 x 10(4)), B2 (Mr 3.0 x 10(4)), C (Mr 1.7 x 10(4)). The subunits saturate anti-TPA serum indistinguishably from TPA. Amino acid composition of TPA and subunits is dominated by glutamic acid, aspartic acid and leucine, cysteine being absent in subunit B1. The isoelectric point of the main subunit, B1, is 4.4-4.6. Sedimentation and diffusion analyses indicate that pure subunit B1 in aqueous solution exists in distinct oligomeric states.

Antigenic properties of the second loop of transforming growth factor alpha by synthetic peptides.

We describe synthesis and immunological properties of peptides constructed based on the second loop of TGF-alpha. The peptides were synthesized manually using a standard solid-phase Boc protocol. Sulfhydryl groups were protected as acetamidomethyl derivatives during the synthesis and the disulfide bridges were formed at high dilution by oxidation with iodine in the presence of glutathione. In ELISA assays antibodies to the peptides were inhibited by the native hormone to various extents. In addition, two peptides reacted well with a polyclonal antibody to TGF-alpha, but this reaction was only slightly inhibited by TGF-alpha in solution.

Synthesis of p-phosphonomethyl-L-phenylalanine using camphor sultam or D-valine as chiral auxiliaries and its incorporation into integrin sequences.

The synthesis of N-Boc-p-phosphonomethyl-L-phenylalanine with two different chiral auxiliaries, camphor sultam or D-valine is described. The preparations have essentially identical properties and have been used to incorporate the amino acid into two integrin peptides as non-hydrolyzable isosteres of phosphotyrosine.

Synthesis and function of an O-phosphorylated peptide corresponding to the cell adhesion sequence of bone sialoprotein (BSP).

Bone sialoprotein contains a cell-binding RGD sequence followed by a threonine residue. Since the protein is extensively phosphorylated, this threonine may also be modified. To study whether such a phosphorylation may alter cell-binding properties, the hexapeptide Pro-Arg-Gly-Asp-Thr(O-phosphoryl)-Tyr has been synthesized by the Fmoc technique using benzyl protective groups for Tyr and phosphate, tert-butyl ester for Asp and Pmc for Arg. Removal of Fmoc groups was effected by treatment with 20% morpholine in DMF. The phospho-peptide inhibited binding of R1 cells to BSP-coated surfaces 10 times less efficiently compared with the non-phosphorylated peptide, as did, surprisingly, also the fibronectin-derived peptide Gly-Arg-Gly-Asp-Ser-Pro.

Synthesis, NMR spectra and function of peptides with alpha-methylserine attached to the RGD sequence of osteopontin.

The three-dimensional structure of the RGD-adhesion sequence has been studied previously by means of linear and cyclic peptides. These peptides show widely differing affinities to integrins, ascribed to a strong dependence on steric factors in the receptor recognition. Insertion of alpha-methylserine next to the RGD sequence in this investigation resulted in lower affinity for both stereoisomers, and several small changes in chemical shifts and coupling constants relative to the parent serine peptide GRGDSL.

An epitope in Coil 2B of cytokeratin 8.

An immunological epitope has been located at the well preserved heptade discontinuity in Coil 2B of human cytokeratin 8, with the aid of synthetic peptides, antibodies to these and monoclonal antibodies to cytokeratins. CD revealed 37% alpha-helix in a 31-peptide.

Synthesis of mono- and disaccharide amino-acid derivatives for use in solid phase peptide synthesis.

N-Fluorenylmethyloxycarbonyl-protected serine and threonine derivatives, carrying O-glycosidically alpha- or beta-linked peracetylated beta-D-Galp-(1-3)-D-GalNAcp carbohydrate chains, were prepared. These derivatives are intended for use in solid phase glycopeptide synthesis. Suitably protected mono- and disaccharide thioglycosides were used as carbohydrate intermediates. These were activated by treatment with bromine to give the glycosyl bromides, which were then used in silver triflate-promoted glycosidations of N-fluorenylmethyloxycarbonyl amino-acid phenacyl esters. Removal of the phenacyl esters with zinc gave the target free acids.

Chemical studies of tissue polypeptide antigen (TPA). II. Partial amino acid sequences of cyanogen bromide fragments of TPA subunit B1.

A subunit (designated B1) of the tumor associated peptide "Tissue Polypeptide Antigen" (TPA) has been cleaved by cyanogen bromide. Some of the fragments contained TPA-activity (as measured by a standardized hemagglutination inhibition method). The fragments have been separated, and partial amino acid sequences have been determined for some of the fragments. A method for purification of the most active fragment (designated BrCN:C) has been worked out from cleavage products obtained by cleavage with cyanogen bromide of "Washed Tissue Powder". By this method fragment BrCN:C has been isolated from a pool of placentae and a pool or carcinoma tumors, and the amino acid sequences of more than 50 amino acids from the N-terminus have been determined. Fragment BrCN:C has been demonstrated from three different sources, which indicates the presence of a common part in subunit B1 independent of source.

Chemical studies of tissue polypeptide antigen (TPA). III. on the nature of the antigenic determinant(s) of TPA subfraction B1.

Tissue Polypeptide Antigen (TPA)*** which is a protein isolated from e.g. human carcinoma cells, has previously been separated into subfractions and studied with biochemical methods. Gel diffusion studies show that the antigenic determinants are retained through the isolation and purification procedures. Specific modifications of the amino acid residues lysine, tyrosine, trytophan and arginine in subfraction B1 have been related to the change in the capacity of the antigen to bind to horse anti-HeLa serum. Complete although reversible loss of binding capacity resulted from blocking of arginine and a minor loss was noted upon modification of tyrosine. No measurable influence was noted upon modification of lysine or tryptophan. No cysteine has been detected in subfraction B1. Circular dichroism measurements show that TPA subfraction B1 is largely alpha-helical in solution, and that no correlation could be detected between antigenic activity and conformation.

Synthesis, NMR and function of an O-phosphorylated peptide, comprising the RGD-adhesion sequence of osteopontin.

The bone phosphoprotein osteopontin owes its cell adhesion property to the RGD-sequence. In order to determine whether a phosphate substituent on the serine following the RGD-sequence interferes with cell binding, we have synthesized GRGDSL along with the corresponding peptide phosphorylated on serine. The latter peptide showed significantly lower cell binding as measured by inhibition of adhesion of R1 cells to surfaces coated with BSP. GRGDSL and phosphorylated GRGDSL show NMR spectra which resemble each other more than that of GRGDSP derived from the fibronectin sequence.

Solid phase synthesis of two phosphorylated peptides related to casein using allyl phosphate protection, and their CD spectra.

Phosphoserine peptides related to the casein kinase II substrate consensus sequence ESLSSSEE have been synthesized using N-Boc-O-diallylphosphono-L-serine by solid-phase methods. The allyl groups were removed, while the peptide was still attached to the solid support, by Pd0 with azide as the nucleophile. Far UV circular dichroism measurements indicate that peptides phosphorylated at Ser 4 or Ser 5 have a somewhat higher content of ordered structure than the non-phosphorylated peptide.

Solid phase synthesis of the fibronectin glycopeptide V(Gal beta 3GalNAc alpha)THPGY, its beta analogue, and the corresponding unglycosylated peptide.

The fibronectin fragment VTHPGY and the corresponding glycopeptides V(Gal beta 3GalNAc alpha)THPGY and V(Gal beta 3GalNAc beta)THPGY were synthesized by the FMOC/solid phase approach. FMOC derivatives of threonine, carrying O-linked, peracetylated Gal beta 3GalNAc chains were used for introduction (HOBt-mediated coupling) of the disaccharide moieties.

Tissue polypeptide antigen (TPA) is related to the non-epidermal keratins 8, 18 and 19 typical of simple and non-squamous epithelia: re-evaluation of a human tumor marker.

Because of the broad clinical interest which tissue polypeptide antigen (TPA) has attracted as a tumor marker, human cell lines and human tissues have been analyzed for TPA expression using immunofluorescence microscopy. Epithelial cell lines including HeLa, MCF-7, and A-431 are recognized by TPA antibodies whereas human lines of non-epithelial origin are not. The positive staining patterns coincide with keratin-type intermediate filaments of the cytoskeleton. On tissue sections a subset of epithelial cells including uterine epithelium, bile duct cells in liver and tumor cells in breast carcinoma are strongly positive; cells of the squamous epithelia of skin and tongue as well as cells of non-epithelial origin are negative. In immunoblots of human epidermis, human tongue mucosa, human hair follicles, Detroit 562 cells, HeLa cells, MCF-7 and RT-4 cells, only keratins 8, 18 and 19 show TPA antigenicity. Conversely a TPA preparation is recognized by various antibodies known to react with keratins, including alpha-IFA, KG 8.13.2 and two antibodies which recognize keratins 18 (CK2) and 19, respectively. Our results thus relate TPA to human keratins 8, 18 and 19 which are known cytoskeletal components in both normal and malignant epithelial cells of simple and non-squamous origin. We speculate that the elevated levels of circulating TPA antigenicity present in the sera of patients with carcinoma, which are often used to monitor tumor progression, correspond to soluble proteolytic fragments originating from this particular keratin subgroup.