Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. Isolation, purification, and core protein characterization

Journal Article · Tue Apr 05 00:00:00 EDT 1988 · J. Biol. Chem.; (United States)

OSTI ID:7143682

Heremans, A; Cassiman, J J; Van den Berghe, H; David, G

Confluent cultured human lung fibroblasts were labeled with 35SO4(2-). After 48 h of labeling, the pericellular matrix was prepared by Triton X-100 and deoxycholate extraction of the monolayers. Heparan sulfate proteoglycan (HSPG) accounted for nearly 80% of the total matrix (35S)proteoglycans. After solubilization in 6 M guanidinium HCl and cesium chloride density gradient centrifugation, the majority (78%) of these (35S) HSPG equilibrated at an average buoyant density of 1.35 g/ml. This major HSPG fraction was purified by ion-exchange chromatography on Mono Q and by gel filtration on Sepharose CL-4B, and further characterized by gel electrophoresis and immunoblotting. Intact (35S)HSPG eluted with Kav 0.1 from Sepharose CL-4B, whereas the protein-free (35S)heparan sulfate chains, obtained by alkaline borohydride treatment of the proteoglycan fractions, eluted with Kav 0.45 (Mr approximately 72,000). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, core (protein) preparations, obtained by heparitinase digestion of 125I-labeled HSPG fractions, yielded one major labeled band with apparent molecular mass of approximately 300 kDa. Reduction with beta-mercaptoethanol slightly increased the apparent Mr of the labeled band, suggesting a single polypeptide structure and the presence of intrachain disulfide bonds. Immunoadsorption experiments and immunostaining of electrophoretically separated heparitinase-digested core proteins with monoclonal antibodies raised against matrix and cell surface-associated HSPG suggested that the major matrix-associated HSPG of cultured human lung fibroblasts is distinct from the HSPG that are anchored in the membranes of these cells. Binding studies suggested that this matrix HSPG interacts with several matrix components, both through its glycosaminoglycan chains and through its heparitinase-resistant core. (Abstract Truncated)

• Research Organization: Univ. of Leuven, Campus Gasthuisberg OandN (Belgium)
• OSTI ID: 7143682
• Journal Information: J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 263:10; ISSN JBCHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINES
ANIMAL CELLS
ANIMALS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CARBOHYDRATES
CENTRIFUGATION
CHEMICAL COMPOSITION
CHROMATOGRAPHY
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
ELECTROPHORESIS
EVEN-ODD NUCLEI
FIBROBLASTS
FRACTIONATION
GLUCOPROTEINS
ION EXCHANGE CHROMATOGRAPHY
ISOTOPES
LIGHT NUCLEI
LUNGS
MAMMALS
MAN
MOLECULAR STRUCTURE
MOLECULAR WEIGHT
MUCOPOLYSACCHARIDES
NUCLEI
ORGANIC COMPOUNDS
ORGANS
OXYGEN COMPOUNDS
POLYSACCHARIDES
PRIMATES
PROTEINS
RADIOISOTOPES
RESPIRATORY SYSTEM
SACCHARIDES
SEPARATION PROCESSES
SOMATIC CELLS
SULFATES
SULFUR 35
SULFUR COMPOUNDS
SULFUR ISOTOPES
ULTRACENTRIFUGATION
VERTEBRATES