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158


Localization of the cleavage sites on fibronectin following digestion by urokinase

Gold LI; Rostagno A; Frangione B; Passalaris T
Urokinase (u-PA) proteolytically cleaves both human plasma (pFn) and cellular (cFn) dimeric fibronectin (M(r) 440,000) into four major polypeptides of approximately M(r) 210,000, 200,000, 25,000, and 6,000. Amino acid sequence analysis of the polypeptide fragments indicated that the enzymatic cleavage of Fn occurs at two sites: 1) between an arginine/alanine peptide bond located C-terminal to residue 259; this cleavage liberates the N-terminal M(r) 25,000 fragment and the M(r) 210,000 and M(r) 200,000 polypeptides derived from the A and B chains of Fn, respectively; and 2) between an arginine/threonine peptide bond located C-terminal to residue 2,299, thereby yielding an M(r) 6,000 dimeric fragment containing the C-terminal interchain disulfide bonds. Predigestion of Fn with u-PA increased the molecule's vulnerability to further attack by the enzymes plasmin and cathepsin D. These data provide further biochemical evidence for the proteolytic cleavage of fibronectin by plasminogen activators and substantiate that u-PA digestion of Fn may be an initial event in the local degradation of the extracellular matrix by malignant cells, possessing elevated levels of these enzymes
PMID: 1469074
ISSN: 0730-2312
CID: 9532

Epitope map of two polyclonal antibodies that recognize amyloid lesions in patients with Alzheimer's disease

Ghiso J; Wisniewski T; Vidal R; Rostagno A; Frangione B
Two synthetic peptides with sequences identical with those of fragments of the extracellular domain of the Alzheimer's-disease amyloid precursor protein (APP) were used to raise antibodies. SP28 comprises positions 597-624 of the APP695 isoform, whereas SP41 extends towards the N-terminus (amino acids 584-624) and contains the entire SP28 peptide. Using e.l.i.s.a. and inhibition experiments we identified the two beta-turn-containing segments 602-607 and 617-624 as the epitopes recognized by anti-SP41 and anti-SP28 respectively. Both antibodies immunolabelled amyloid lesions in brains from Alzheimer's-disease patients and patients with related disorders, whereas they were unreactive in control brains. However, when probed on immunoblots, anti-SP28 failed to detect full-length APP from baculovirus-infected Sf9 cells, and anti-SP41 reacted weakly compared with other anti-APP antisera. The data suggest that these antibodies are directed to conformational epitopes not existent in the native molecules but present after alternative APP processing
PMCID:1130811
PMID: 1372166
ISSN: 0264-6021
CID: 9414

Biochemical studies on the interaction of fibronectin with Ig

Rostagno AA; Frangione B; Gold L
We have previously biochemically characterized three separate sites on the fibronectin (Fn) molecule that interact with IgG. These studies have been extended to examine the interaction of Fn with other classes and subclasses of Ig. By ELISA, a preferential quantitative binding order of Fn to the major Ig classes and subclasses was obtained as follows: IgG greater than IgM greater than IgA, IgG1 greater than IgG3 = IgG4 greater than IgG2, and IgA1 = IgA2. Using fragments of Fn obtained by subtilisin digestion followed by IgM and IgA affinity chromatography, immunoblot analysis using monospecific antisera to separate regions of the Fn molecule, and amino acid sequence analysis, these studies indicate that polyclonal IgA and IgM interact with Fn in the same three regions and under the same ionic conditions as previously described for IgG. Site 1 is a 22-kDa fragment that commences at residue 1 of the Fn molecule. Sites 2 (16 kDa) and 3 (26-29 kDa) begin at residues 588 and 1597, respectively. Under physiological conditions a monoclonal antibody that recognizes site 1 completely inhibited the interaction of intact Fn with IgG, IgM, and IgA. Therefore, this is the only physiologically active site in the intact molecule. Aggregated but not monomeric IgG competitively inhibited the binding of Fn to IgG-coated microtiter ELISA plates; thus, this interaction can take place in a fluid-phase system. These results indicate that Fn can potentially interact with immune complexes and aggregates of all Ig in the circulation and thus may play a significant role in both their clearance and deposition in Fn-containing tissues, such as occurs in immune-complex-related disorders
PMID: 2016522
ISSN: 0022-1767
CID: 9547

ALZHEIMER'S AMYLOID PRECURSOR PROTEIN CONTAINS A TETRAPEPTIDE SEQUENCE-RHIDS THAT PROMOTES CELL ADHESION [Meeting Abstract]

GHISO J; ROSTAGNO A; FRANGIONE B
BIOSIS:PREV199242104041
ISSN: 0190-5295
CID: 101623

FURTHER LOCALIZATION OF THE TWO BINDING SITES FOR FIBRIN WITHIN FIBRONECTIN [Meeting Abstract]

GOLD L; ROSTAGNO A
BIOSIS:PREV199140130200
ISSN: 0733-1959
CID: 101624

Binding of cystatin C to C4: the importance of sense-antisense peptides in their interaction

Ghiso J; Saball E; Leoni J; Rostagno A; Frangione B
Hydropathic anticomplementarity of amino acids indicates that peptides derived from complementary DNA strands may form amphiphilic structures and bind one another. By using this concept, we have found that the antisense peptide Ser-Tyr-Asp-Leu complementary to the segment Gln-Ile-Val-Ala-Gly (residues 55-59) in cystatin C (an inhibitor of cysteine proteases) is located at positions 611-614 of the beta chain of human C4, the fourth component of complement. Here we describe and characterize the specific interaction between cystatin C and C4 by ligand affinity chromatography and ELISA. Interaction between the two native proteins was mimicked on replacement of one of them with the corresponding sense-antisense peptide coupled to a carrier protein, and the binding was inhibited by these synthetic peptides in solution. Through the interaction with C4, cystatin C may play a regulatory role in complement activation that might be of particular importance at tissue sites where both proteins are produced by macrophages
PMCID:53459
PMID: 2304899
ISSN: 0027-8424
CID: 9432

Biochemical characterization of the fibronectin binding sites for IgG

Rostagno AA; Frangione B; Gold LI
Plasma fibronectin (Fn) is a constituent of cryoglobulins and has been shown to interact with immune complexes. In a previous report we demonstrated that Fn specifically bound to IgG immobilized on a solid matrix. To localize and biochemically characterize the sites on the Fn molecule involved in this interaction, Fn was enzymatically cleaved with subtilisin and subjected to IgG affinity chromatography. Three major polypeptide fragments of 16 kDa, 22 kDa, and a triplet of 26- to 29-kDa bound IgG. They were localized to three separate regions of the molecule by Western blot analysis using antisera to specific regions of the Fn molecule, by amino acid sequencing, and by their previously described heparin binding affinities. The 22-kDa fragment interacted with IgG under physiologic conditions and it is localized at the N-terminal of the Fn molecule. The 16-kDa and 26- to 29-kDa fragments bound to IgG under conditions of lower ionic strength; the former commences at residue 588, carboxyl-terminal to the collagen binding region and the latter begins at residue 1597, carboxyl-terminal to the cell binding domain. The interaction of Fn with Ig has significant implications in host defense and also in immune complex disease where basement membrane Fn may sequester immune complexes from the circulation
PMID: 2553808
ISSN: 0022-1767
CID: 9560

Affinity of fibronectin for polyclonal IgG

Salvarrey M; Rostagno A
Fibronectin (Fn) is an adhesive glycoprotein that plays an important role in reticuloendothelial system functioning. Fn binds several macromolecules, it is found in cryoprecipitates obtained from patients with different diseases and it interacts in vitro with immune complexes. The interaction between Fn and polyclonal IgG was characterized by ligand affinity chromatography. After IgG was modified by attachment to a solid matrix or heat aggregation, it was able to interact with either soluble or immobilized Fn. The binding was highly influenced by ionic strength and pH. The estimated affinity constants were 3.0 x 10(6)/M when soluble Fn was applied to solid phase IgG and 2.3 x 10(7)/M when aggregated IgG interacted with immobilized Fn. The interaction may be relevant in situations in which immune complexes are involved
PMCID:1541747
PMID: 2736803
ISSN: 0009-9104
CID: 56306

Fibronectin : biochemical characterization of the binding to C3. Effect of chemical modifications on the interaction [Meeting Abstract]

Saball DE; Rostagno A
ORIGINAL:0006197
ISSN: 0025-7680
CID: 73973

BIOCHEMICAL CHARACTERIZATION OF UROKINASE PROTEOLYTIC CLEAVAGE OF FIBRONECTIN

GOLD L I; ROSTAGNO A; PASSALARIS T
BIOSIS:PREV198936082820
ISSN: 0021-9525
CID: 101625