Glycosaminoglycans Differentially Bind HARP and Modulate Its Biological Activity

Abstract

Heparin affin regulatory peptide (HARP) is a polypep-) and a lower affinity (K d ‫؍‬ 51 nM). Exogenous heparin, heparan sulfate, and dermatan sulfate potentiated the growth-stimulatory activity of HARP, suggesting that corresponding proteoglycans could be involved in the regulation of the mitogenic activity of HARP. Heparin affin regulatory peptide (HARP)1 belongs to a growing group of heparin binding extracellular regulatory molecules, and it has mitogenic and neurite outgrowth activities.Independently purified from perinatal rat brain as a polypeptide that induces neurite outgrowth of embryonic neurons (1), this protein, also named pleiotrophin (2), has been purified from uterus (3) and adult brain (4) as a growth factor for fibroblastic and endothelial cells. Expression of HARP mRNA is developmentally regulated, and major expression is found during the perinatal growth period in rat brain (5, 6), pointing to the possible function of this molecule in the maturation of nerve cells. Moreover, HARP mRNA is found in several adult tissues, and HARP has been implicated in tumor growth (7).HARP cDNA has been cloned and sequenced from several species. The predicted amino acid sequence has been determined in humans, mice, and rats (2, 6, 8, 9) and shows 98% homology among the three species. In addition, the HARP amino acid sequence shares 55% homology with the midkine gene product (10). In contrast to their neurite outgrowth activity, initial studies differed in their results about the mitogenic activity of midkine and HARP (3, 9, 11-15), possibly because of differences in the cell type used or to the isolation procedure.More recent studies clearly demonstrate that this family of polypeptides have growth-stimulatory activity (16).The high affinity of HARP for heparin suggests that HARP may bind to heparan sulfate proteoglycans (HSPGs) present in extracellular compartments defined as cell surface and extracellular matrix (ECM). Recent studies have demonstrated that syndecan-1, syndecan-3 (N-syndecan), and syndecan-4 (ryudocan) bind HARP with high affinity (13,17,18). In addition, biochemical and cell biological studies have pointed to syndecan-3 as the HARP receptor involved in neurite outgrowth activity (19). Despite the correlation derived from previous studies, there has been no direct biochemical demonstration that HARP is trapped in the extracellular compartment as a mitogenic molecule bound to HSPGs. In this study we investigated whether HARP is present in the extracellular compartment as well as the role of glycosaminoglycans (GAGs) in controlling HARP mitogenic activity. (Proteus vulgaris; EC 4.2.2.4), leupeptin, pepstatin, phenylmethylsulfonyl fluoride, heparan sulfate (HS) from bovine intestinal mucosa, keratan sulfate (KS) from bovine cornea, chondroitin sulfate A (CS-A) from bovine trachea, dermatan sulfate (DS) from porcine skin, and chondroitin sulfate C (CS-C) from shark cartilage were purchased from Sigma. Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulins were obtained from Diagnostics-...

Dominant Negative Effectors of Heparin Affin Regulatory Peptide (HARP) Angiogenic and Transforming Activities

“…Each ␤-sheet domain contains a heparin-binding site, which plays a role in the modulation of HARP mitogenic activity (20). In addition, we have shown that at least one heparin-binding domain is involved in the dimerization of HARP (21).…”

16-kDa fragment of pleiotrophin acts on endothelial and breast tumor cells and inhibits tumor development

“…Western Blot Analysis and ELISA The PTN expression in supernatants, cell pellets, and extracellular compartments defined as cell surface and extracellular matrix MDA-MB-231 tumors was done in MDA-MB-231 and PC3 cells by Western blot analysis following heparin Sepharose precipitation as described previously (20). Quantification of the immunoreactive material was done by densitometric analysis using a ChemiGenius system and the Genetools software (Syngene).…”

Structural and Functional Characterization of Oversulfated Chondroitin Sulfate/Dermatan Sulfate Hybrid Chains from the Notochord of Hagfish

“…DS from porcine skin shows negligible binding to immobilized MK compared with CS-H (12), although the 3 M NaCl-eluted fraction was used previously. On the other hand, DS from porcine skin binds PTN with a K d of 51 nM when DS is immobilized on the sensor's surface (39).…”