Hyaluronic acid-receptor binding demonstrated by synthetic adhesive proteoglycan peptide constructs and by cell receptors on the marine sponge Microciona prolifera.
Synthetic peptide constructs of RHAMM isoforms were prepared as described by Zhang et al. (10). The putative HA binding sequence of MAF core protein was synthesized in the Biotechnology Laboratories at the University of Toronto. This thirteen amino acid construct possessed short sequences that flanked the eight primarily basic amino acids believed to possess HA binding affinities. The structure was as follows: GLY-VAL-SER-VAL-ARG-ARG-TYR-ARGASN-ARG-VAL-ARG-ILE-OH. Dot blots were carried out by spotting 10 micrograms of a peptide at a site on nitrocellulose. After blocking non-specific reactive sites with 1% buffered bovine serum albumin, BHA was applied and, after incubation and washings, was followed in sequence by streptavidin peroxidase and color development with diaminobenzidine. The negative controls consisted of peptide-free MAF glycans and a peptide-free color development site [ILLUSTRATION FOR FIGURE 1E OMITTED].
The reactivities of cellular RHAMM sites toward specific anti-RHAMM antibodies and HA were studied comparatively by immunohistochemical experiments in which sponge cells were double-labeled with mouse anti-RHAMM antibodies and with BHA. Sections of formalin-fixed and paraffin-embedded sponge cell pellets, cut at 5 [[micro]meter], were deparaffinated and were double-labeled with: (a) BHA stain as the first reagent, followed by mouse anti-RHAMM and FITC anti-mouse antibodies; and (b) anti-RHAMM FITC anti-mouse as the first reagent, followed by BHA and color development. Stained sections were viewed under tungsten and UV light.
The presence of color development at all sites that were blotted with RHAMM peptide and MAF peptide constructs indicated that they were reactive with hyaluronic acid (BHA). On the other hand, there was no demonstrable staining at a control site treated with peptide-free MAF, or at a site lacking MAF and peptide. The staining patterns shown on microscopic sections indicated that the reactivity of BHA toward RHAMM sites could be blocked by the prior application of anti-RHAMM antibodies [ILLUSTRATION FOR FIGURE 1C, D OMITTED], in which case some immunofluorescence by the anti-RHAMM FITC preparation could be shown. Conversely, the activity of antiRHAMM toward RHAMM foci was greatly reduced when the BHA stain had been applied beforehand [ILLUSTRATION FOR FIGURE 1A, B OMITTED]. This indicated that either one or the other reagent could compete effectively for cellular RHAMM based upon its ability to block the reaction of the alternate probe.
Taken together, the evidence indicates that (a) the postulated HA binding sequence of MAF core peptide actually functions in this role as determined by the behavior of an HA binding mimetic, and (b) this MAF HA binding sequence, in addition to the RHAMM on Microciona cell membranes, may serve as anchorage points for HA molecules. In this role, the entire construct may provide a conduit for the transduction of signals that specify motility and perhaps other phenomena. As a final point, sponges like Microciona are the most primitive species with multiple cell lineages, in which cell-cell recognition occurs by clumping. (11). The presence of HA receptors on MAF and on sponge cells suggests a highly conserved function that may possess a specific role in motility. This function may thus help to bring about cell sorting and aggregation, in synergy with other MAF-binding epitopes that have been referred to in earlier publications (12-15).
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