Binding site in human plasma fibronectin to HL-60 cells localizes in the C-terminal heparin-binding region independently of RGD and CS1.
A 29-kDa monomeric dispase-digestive fragment of human plasma fibronectin has been purified by heparin affinity chromatography. The NH2-terminal sequence was determined as Ala1687-Val-Thr-Thr-Ile-Pro-Ala-Pro. By mass spectrometry the molecular weight was determined to be 30,241.9 with standard deviation of 3.9 amu. Therefore, we defined the C-terminal sequence of the 29-kDa fragment as Arg1957-Lys-Lys-Thr-Gly-Gln-Glu. This indicates that the fragment is composed of 277 amino acids. 125I-fibronectin and the 125I-labeled 29-kDa fragment bound to HL-60 (human acute promyelocytic leukemia) cells in a time-dependent, saturable, and reversible manner. Approximately 120 min was required to reach maximal binding. There were no differences in quantity or rate of binding of labeled fibronectin and 29-kDa fragment at temperatures of 4 degrees, 22 degrees, and 37 degrees C. The number of binding sites per HL-60 cell of fibronectin and the 29-kDa fragment were 140,000 with a Kd of 133 nM and 108,000 with a Kd of 250 nM, respectively. The binding of fibronectin to HL-60 cells was completely inhibited by this fragment, and by the peptides of RGDS and CS1 with IC50s of 3.6, 840, and 670 microM, respectively. Native fibronectin inhibited the direct binding of the 29-kDa fragment to HL-60 cells; however, RGDS peptide, peptide CS1, or two melanoma cell adhesion-promoting domain peptides in this 29-kDa fragment (peptide I; Tyr1906-Val1924, peptide II; Asp1946-Thr1960) did not block this binding. Neither heparitinase nor chondroitinase treatment of cells had any effect on these bindings. These results indicate that the C-terminal cell- and heparin-binding domain of fibronectin mediates HL-60 cell binding by direct interaction independently of RGD, CS1, and melanoma cell adhesion domains in this fragment.
Fujita
H
H
First Department of Internal Medicine, Yokohama City University School of Medicine.
Mohri
H
H
Kanamori
H
H
Iwamatsu
A
A
Okubo
T
T
D000595:Amino Acid Sequence; D001665:Binding Sites; D005353:Fibronectins / Q000097:blood / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D011509:Proteoglycans / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014407:Tumor Cells, Cultured
D005353:Fibronectins; D036341:Intercellular Signaling Peptides and Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D011509:Proteoglycans; C076687:CS1 peptide; C047981:arginyl-glycyl-aspartic acid; D006493:Heparin; C059453:RGES peptide; C046617:arginyl-glycyl-aspartyl-serine