Abstract
A high molecular weight basement membrane heparan sulfate proteoglycan, isolated from murine Englebreth-Holm-Swarm tumor, is seen in platinum replicas as an elongated flexible core (Mr = 450,000) consisting of a series of tandem globular domains from which extend, at one end, two to three heparan sulfate chains (average Mr = 80,000 each). This macromolecule will self-assemble into dimers and lesser amounts of oligomers when incubated in neutral isotonic buffer. These molecular species can be separated by zonal velocity sedimentation and assembly is seen to be time- and concentration-dependent. In rotary-shadowed platinum replicas the binding region is found at or near the end of the core at the pole opposite the origin of the heparan sulfate chains. Dimers are double-length structures and oligomers are seen as stellate clusters: in both, the heparan sulfate chains appear peripherally oriented. While isolated cores self-assemble, isolated heparan sulfate chains do not bind intact proteoglycans. Furthermore, proteolytic removal of a non-heparan sulfate containing core moiety destroys the ability of the proteoglycan monomer to form larger species or bind intact proteoglycan, further supporting the binding topography determined morphologically. These negatively charged macromolecular complexes may be important contributors to basement membrane structure and function.
Highlights
Evaluation of Proteoglycan Fragments for Self-msemblyIsolated core protein(prepared by heparitinase digestion) followingincubation at 37 "Cwas noted in Pt/C replicas (Fig. 8) to be distributed as monomers, dimers, and oligomers without theirthread-like extensions
Upon incubation at 37 "C for several hours, the proteoglycan remained as a single peak (Fig. 9C) unchanged in its sedimentation characteristics and larger complexes were not seen in rotary-shadowed replicas
When the assembly behavior of the tryptic fragment was compared to intact proteoglycan at a lower temperature (28 "C) for less time (3-4 h), similar results were obtained in which the tryptic fragment remained as a single 7 S peak
Summary
Proteolytic removal of a non-heparan sulfate containing core moiety destroys the ability of the proteoglycan monomer to form larger species or bind intact proteoglycan, further supporting the binding topography determined morphologically These negatively charged macromolecularcomplexes may be important contributors to basement membrane structure and function. While the low molecular weight form is extractable with 0.5 M NaCl and appears to have only weak associations with other matrix components [6], the high molecular weight form can only be extracted with dissociative concentrations of urea or guanidine and probably has significant interactions with other basement membrane structural components This heparan sulfate proteoglycan has an elongated multidomain core protein consisting of a single polypeptide with M,of. In this studywe describe a novel self-assembly interaction of the high molecular weight form of heparan sulfate proteoglycan in which monomeric macromoleculesbind at theends of their cores to form dimers and stellate oligomers with peripherally oriented glycosaminoglycan chains
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