Ultrastructural localization of the major proteoglycan and type II procollagen in organelles and extracellular matrix of cultured chondroblasts

The mechanisms of synthesis and intracellular routing of the various cartilage matrix macromolecules are still unclear. We have studied this problem in cultured chondroblasts at the ultrastructural level using monospecific antibodies against the core protein of the keratan sulfate/chondroitin sulfate-rich cartilage proteoglycan (KS:CS-PG) or Type II procollagen, and cuprolinic blue, a cationic dye that binds to the glycosaminoglycan chains of proteoglycans. Intracellularly, the proteoglycan antibodies localized KS:CS-PG and its precursors primarily in the Golgi complex and secretory vesicles. In contrast, the bulk of Type II procollagen was found within the rough endoplasmic reticulum (ER). While devoid of collagen, the extracellular matrix was rich in KS:CS-PG molecules some of which studded the chondroblast plasmalemma. Cuprolinic blue staining indicated that the proteoglycans present in the Golgi complex fell into a predominant class of large proteoglycans, probably representing KS:CS-PG, and a minor class of smaller proteoglycans. Groups of these divergent proteoglycans often occupied distinct Golgi subcompartments; moreover, single large proteoglycans appeared to align along the luminal surface of Golgi cisternae and secretory vesicles. These results suggest that in cultured chondroblasts KS:CS-PG and Type II procollagen are differentially distributed both in organelles and in the extracellular matrix, and that different proteoglycan types may occupy distinct subcompartments in trans Golgi.     

Chondroitin sulfate proteoglycan is a constituent of the basement membrane in the rat embryo parietal yolk sac

Summary In addition to containing Type IV collagen, laminin and entactin, basement membranes contain small amounts of proteoglycans substituted primarily with heparan sulfate chains. We have previously shown, however, that parietal yolk sacs in organ culture synthesize predominantly chondroitin sulfate proteoglycan. In the present study, we have used histochemical and immunohistochemical techniques coupled with chondroitinase ABC digestion to provide evidence for the presence of chondroitin sulfate proteoglycan in the basement membrane (Reichert's membrane) of the 14.5-day rat embryo parietal yolk sac. The results revealed numerous cuprolinic blue-positive filaments and granules, 20–30 nm in greater length or diameter, dispersed throughout the thickness of the basement membrane. Both structures were removed by preincubating freshly isolated parietal yolk sacs with chondroitinase ABC. A similar labeling pattern was also obtained with immunoelectron microscopy using gold-labeled monoclonal anti-bodies directed against the three major isomers of protein-bound chondroitin sulfate. In contrast, coarser cuprolinic blue granules, 40–100 nm in diameter, were neither sensitive to chondroitinase ABC digestion nor labeled by the monoclonal antibodies. These results thus indicate that Reichert's membrane contains chondroitin sulfate proteoglycan in addition to heparan sulfate proteoglycan.     

Ultrastructural localization of the major proteoglycan and type II procollagen in organelles and extracellular matrix of cultured chondroblasts

Summary The mechanisms of synthesis and intracellular routing of the various cartilage matrix macromolecules are still unclear. We have studied this problem in cultured chondroblasts at the ultrastructural level using (i) monospecific antibodies against the core protein of the keratan sulfate/chondroitin sulfate-rich cartilage proteoglycan (KS:CS-PG) or Type II procollagen, and (ii) cuprolinic blue, a cationic dye that binds to the glycosaminoglycan chains of proteoglycans. Intracellularly, the proteoglycan antibodies localized KS:CS-PG and its precursors primarily in the Golgi complex and secretory vesicles. In contrast, the bulk of Type II procollagen was found within the rough endoplasmic reticulum (ER). While devoid of collagen, the extracellular matrix was rich in KS:CS-PG molecules some of which studded the chondroblast plasmalemma. Cuprolinic blue staining indicated that the proteoglycans present in the Golgi complex fell into a predominant class of large proteoglycans, probably representing KS:CS-PG, and a minor class of smaller proteoglycans. Groups of these divergent proteoglycans often occupied distinct Golgi subcompartments; moreover, single large proteoglycans appeared to align along the luminal surface of Golgi cisternae and secretory vesicles. These results suggest that in cultured chondroblasts KS:CS-PG and Type II procollagen are differentially distributed both in organelles and in the extracellular matrix, and that different proteoglycan types may occupy distinct subcompartments in trans Golgi.     

Basement membrane proteoglycans: Modulators <Emphasis Type="Italic">Par Excellence</Emphasis> of cancer growth and angiogenesis

Proteoglycans located in basement membranes, the nanostructures underling epithelial and endothelial layers, are unique in several respects. They are usually large, elongated molecules with a collage of domains that share structural and functional homology with numerous extracellular matrix proteins, growth factors and surface receptors. They mainly carry heparan sulfate side chains and these contribute not only to storing and preserving the biological activity of various heparan sulfate-binding cytokines and growth factors, but also in presenting them in a more Ȍactive configurationȍ to their cognate receptors. Abnormal expression or deregulated function of these proteoglycans affect cancer and angiogenesis, and are critical for the evolution of the tumor microenvironment. This review will focus on the functional roles of the major heparan sulfate proteoglycans from basement membrane zones: perlecan, agrin and collagen XVIII, and on their roles in modulating cancer growth and angiogenesis.