Modulation of triple-helical stability and subsequent melanoma cellular responses by single-site substitution of fluoroproline derivatives.
Collagen is a multifunctional protein, serving as a structural scaffold and a modulator of cellular responses. Prior work has identified distinct regions from several collagen types that promote cell adhesion, spreading, migration, and signal transduction. One of these regions, alpha1(IV)1263-1277 from type IV collagen, mediates these responses via melanoma cell CD44-chondrotin sulfate proteoglycan receptors. In the study presented here, we have used a triple-helical model of alpha1(IV)1263-1277 to evaluate (a) conformational stability and (b) cellular responses based on single-site incorporation of trans-4-fluoro-L-proline (trans-Flp) or cis-4-fluoro-L-proline (cis-Flp) for trans-4-hydroxy-L-proline (trans-Hyp). The structural effects of cis-Flp and trans-Flp substitution were studied by circular dichroism and NMR spectroscopies. The peptide containing a single trans-Flp instead of trans-Hyp was slightly more thermally stable than the parent peptide (T(m) = 37 vs 34 degrees C), while the peptide containing cis-Flp was considerably less stable than the parent peptide (T(m) = 30 degrees C). Melanoma cell adhesion and spreading were examined under conditions where the trans-Hyp-, trans-Flp-, and cis-Flp-containing ligands were approximately 15, <10, and approximately 65% denatured, respectively. Adhesion to each of the three ligands was remarkably sensitive to the respective ligand conformation, with EC(50) values of approximately 2.5, approximately 0.35, and >5.0 microM for the trans-Hyp-, trans-Flp-, and cis-Flp-containing ligands, respectively. Melanoma cell spreading was quantitated over a ligand concentration range of 0.01-50 microM and, in a fashion similar to adhesion, was more extensive on the trans-Flp ligand than on the trans-Hyp ligand. Very low levels of spreading were observed with the cis-Flp-containing ligand at all concentrations tested. Melanoma cell adhesion to and spreading on the three ligands suggested the dramatic biological consequence of even subtle changes in relative triple-helical content. Such subtle changes may model those occurring in the basement membrane during the tumor cell invasion process, and thus provide mechanistic insight into this stage of metastasis.
Malkar
Navdeep B
NB
Department of Chemistry and Biochemistry, Florida Atlantic University, 777 Glades Road, Boca Raton, Florida 33431-0991, USA.
Lauer-Fields
Janelle L
JL
Borgia
Jeffrey A
JA
Fields
Gregg B
GB
D000595:Amino Acid Sequence; D001485:Basement Membrane / Q000737:chemistry; D055592:Biophysical Phenomena; D001703:Biophysics; D002448:Cell Adhesion; D024141:Collagen Type IV / Q000737:chemistry* / Q000502:physiology; D004355:Drug Stability; D006801:Humans; D008024:Ligands; D008545:Melanoma / Q000737:chemistry* / Q000503:physiopathology*; D008969:Molecular Sequence Data; D009361:Neoplasm Invasiveness; D011392:Proline / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D017433:Protein Structure, Secondary; D013237:Stereoisomerism; D014407:Tumor Cells, Cultured
D024141:Collagen Type IV; D008024:Ligands; C431803:fluoro-proline; D011392:Proline