Reconstitution of high cell binding affinity of a marine sponge aggregation factor by cross-linking of small low affinity fragments into a large polyvalent polymer.
Species-specific reaggregation of cells from the marine sponge Microciona prolifera is mediated by a proteoglycan-like aggregation factor (MAF) of Mr = 2 X 10(7) which has two functional domains, a cell binding domain and an aggregation factor interaction domain. After extensive trypsin digestion, over 60% of the MAF mass was converted into a glycopeptide fragment of Mr = 10,000 (T-10) which is therefore a representative part of the major portion, but not of the entire MAF molecule. The T-10 fragment has a similar amino acid and carbohydrate composition as the intact MAF and displays species-specific binding. Although T-10 also inhibited MAF association with homotypic cells, its apparent affinity is 3 X 10(6) M-1, i.e. 13,000 times lower than that of native MAF. Reconstitution of binding affinity in the same order of magnitude as native MAF (Ka = 10(10) M-1) was obtained by cross-linking the glycopeptide fragment into polymers of the approximate size of MAF (Mr greater than 1.5 X 10(7) using diepoxybutane and glutaraldehyde, or periodate oxidation and glutaraldehyde. The apparent association constants of intermediate polymers with Mr = 1 X 10(5), 6 X 10(5), 9 X 10(5), 2 X 10(6) and above 1.5 X 10(7) increased proportionally to their size and were in line with association constants of MAF degradation fragments. Since the binding affinity of the T-10 glycopeptide fragment could be reconstituted by cross-linking, and since this fragment accounts for over 60% of MAF, we propose that the specificity and high affinity of the MAF-cell association is based on a highly polyvalent interaction of low affinity cell-binding sites. Such a polyvalency of the cell binding domain is advantageous for efficient cell-cell interactions and thus differs from most known interaction molecules and receptors characterized.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D015815:Cell Adhesion Molecules; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D011108:Polymers / Q000378:metabolism; D011161:Porifera / Q000032:analysis*; D011506:Proteins / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D002241:Carbohydrates; D015815:Cell Adhesion Molecules; D011108:Polymers; D011506:Proteins; C013065:cell aggregation factors; D014357:Trypsin