Analysis of the proteoglycans synthesized by human bone cells in vitro

J. N. Beresford, Neal S Fedarko, L. W. Fisher, R. J. Midura, M. Yanagishita, J. D. Termine, P. G. Robey

Research output: Contribution to journalArticle

Abstract

Proteoglycans were isolated by ion-exchange chromatography from the extracted cell layer and culture medium of human bone cell cultures following incubation in the presence of [35S]sulfate and [3H]leucine. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the synthesized proteoglycans consisted of at least five polydisperse species having median apparent M(r) = 600,000, 400,00, 270,000, 135,000 and 40,000. When chromatographed further on octyl-Sepharose CL-4B, the proteoglycans of the cell layer resolved into three peaks. The unbound fraction (peak cell layer-I) contained a 40,000 species consisting of a single glycosaminoglycan chain with or without peptide. Peak cell layer-II contained three sulfated species on electrophoresis: a 600,000 species uniformly distributed across the peak, a 135,000 species enriched in the ascending limb (similar to bone PG-I as described previously), and a 270,000 species (similar to bone PG-I) enriched in the descending limb. Peak cell layer-III, eluting at 0.2% Triton X-100, was highly enriched in a 400,000 proteoglycan component. When media proteoglycans were chromatographed on octyl-Sepharose, two labeled peaks were found. Peak medium-I (unbound) contained a species exhibiting electrophoretic mobility similar to that of the 400,000 species present in peak cell layer-III. Peak II of the culture medium (medium-II) was apparently identical to that of peak cell layer-II, containing the 600,000, 270,000 and 135,000 species. No appreciable 40,000 species was observed in the medium. Treatment of the 600,000 species with either chondroitinase ABC or ACII generated a core protein preparation with bands of 390,000 and 340,000 on SDS gels. Neither the intact nor the chondroitinase ABC-treated 600,000 species was immunoprecipitated by a purified, polyclonal antiserum raised against the core protein of the large chondroitin sulfate proteoglycan of human articular cartilage. Treatment of the 270,000 and 135,000 proteoglycans with chondroitinase ABC, but not ACII, generated a core protein preparation with bands of 52,000 and 49,000 on SDS gels, indicating that they were dermatan sulfate-containing species. The 400,000 species contained both heparan sulfate and chondroitin sulfate, in approximately a 3:1 labeling ratio. This species changed in electrophoretic mobility following treatment with chondroitinase ABC, heparatinase, or both enzymes in combination, which suggested that it may be a hybrid proteoglycan (i.e. both types of glycosaminoglycan chain on the same core protein). A polyclonal antiserum raised against a mixture of the small proteoglycans purified from the mineral compartment of human bone, immunoprecipitated the intact and chondroitinase ABC-treated 270,000 and 135,000 bone cell proteoglycans. This same antiserum failed to immunoprecipitate either the intact or chondroitinase ABC-treated 600,000 species, indicating that the core proteins prepared from the 270,000 and 135,000 species are antigenically distinct from those of the 600,000 species.

Original languageEnglish (US)
Pages (from-to)17164-17172
Number of pages9
JournalJournal of Biological Chemistry
Volume262
Issue number35
StatePublished - 1987
Externally publishedYes

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Proteoglycans
Chondroitin ABC Lyase
Bone
Bone and Bones
Cells
Immune Sera
Electrophoretic mobility
Glycosaminoglycans
Proteins
Electrophoresis
Culture Media
Extremities
Cell Culture Techniques
Gels
Chondroitin Sulfate Proteoglycans
In Vitro Techniques
Dermatan Sulfate
Heparitin Sulfate
Chondroitin Sulfates
Ion Exchange Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Beresford, J. N., Fedarko, N. S., Fisher, L. W., Midura, R. J., Yanagishita, M., Termine, J. D., & Robey, P. G. (1987). Analysis of the proteoglycans synthesized by human bone cells in vitro. Journal of Biological Chemistry, 262(35), 17164-17172.

Analysis of the proteoglycans synthesized by human bone cells in vitro. / Beresford, J. N.; Fedarko, Neal S; Fisher, L. W.; Midura, R. J.; Yanagishita, M.; Termine, J. D.; Robey, P. G.

In: Journal of Biological Chemistry, Vol. 262, No. 35, 1987, p. 17164-17172.

Research output: Contribution to journalArticle

Beresford, JN, Fedarko, NS, Fisher, LW, Midura, RJ, Yanagishita, M, Termine, JD & Robey, PG 1987, 'Analysis of the proteoglycans synthesized by human bone cells in vitro', Journal of Biological Chemistry, vol. 262, no. 35, pp. 17164-17172.
Beresford JN, Fedarko NS, Fisher LW, Midura RJ, Yanagishita M, Termine JD et al. Analysis of the proteoglycans synthesized by human bone cells in vitro. Journal of Biological Chemistry. 1987;262(35):17164-17172.
Beresford, J. N. ; Fedarko, Neal S ; Fisher, L. W. ; Midura, R. J. ; Yanagishita, M. ; Termine, J. D. ; Robey, P. G. / Analysis of the proteoglycans synthesized by human bone cells in vitro. In: Journal of Biological Chemistry. 1987 ; Vol. 262, No. 35. pp. 17164-17172.
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abstract = "Proteoglycans were isolated by ion-exchange chromatography from the extracted cell layer and culture medium of human bone cell cultures following incubation in the presence of [35S]sulfate and [3H]leucine. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the synthesized proteoglycans consisted of at least five polydisperse species having median apparent M(r) = 600,000, 400,00, 270,000, 135,000 and 40,000. When chromatographed further on octyl-Sepharose CL-4B, the proteoglycans of the cell layer resolved into three peaks. The unbound fraction (peak cell layer-I) contained a 40,000 species consisting of a single glycosaminoglycan chain with or without peptide. Peak cell layer-II contained three sulfated species on electrophoresis: a 600,000 species uniformly distributed across the peak, a 135,000 species enriched in the ascending limb (similar to bone PG-I as described previously), and a 270,000 species (similar to bone PG-I) enriched in the descending limb. Peak cell layer-III, eluting at 0.2{\%} Triton X-100, was highly enriched in a 400,000 proteoglycan component. When media proteoglycans were chromatographed on octyl-Sepharose, two labeled peaks were found. Peak medium-I (unbound) contained a species exhibiting electrophoretic mobility similar to that of the 400,000 species present in peak cell layer-III. Peak II of the culture medium (medium-II) was apparently identical to that of peak cell layer-II, containing the 600,000, 270,000 and 135,000 species. No appreciable 40,000 species was observed in the medium. Treatment of the 600,000 species with either chondroitinase ABC or ACII generated a core protein preparation with bands of 390,000 and 340,000 on SDS gels. Neither the intact nor the chondroitinase ABC-treated 600,000 species was immunoprecipitated by a purified, polyclonal antiserum raised against the core protein of the large chondroitin sulfate proteoglycan of human articular cartilage. Treatment of the 270,000 and 135,000 proteoglycans with chondroitinase ABC, but not ACII, generated a core protein preparation with bands of 52,000 and 49,000 on SDS gels, indicating that they were dermatan sulfate-containing species. The 400,000 species contained both heparan sulfate and chondroitin sulfate, in approximately a 3:1 labeling ratio. This species changed in electrophoretic mobility following treatment with chondroitinase ABC, heparatinase, or both enzymes in combination, which suggested that it may be a hybrid proteoglycan (i.e. both types of glycosaminoglycan chain on the same core protein). A polyclonal antiserum raised against a mixture of the small proteoglycans purified from the mineral compartment of human bone, immunoprecipitated the intact and chondroitinase ABC-treated 270,000 and 135,000 bone cell proteoglycans. This same antiserum failed to immunoprecipitate either the intact or chondroitinase ABC-treated 600,000 species, indicating that the core proteins prepared from the 270,000 and 135,000 species are antigenically distinct from those of the 600,000 species.",
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T1 - Analysis of the proteoglycans synthesized by human bone cells in vitro

AU - Beresford, J. N.

AU - Fedarko, Neal S

AU - Fisher, L. W.

AU - Midura, R. J.

AU - Yanagishita, M.

AU - Termine, J. D.

AU - Robey, P. G.

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N2 - Proteoglycans were isolated by ion-exchange chromatography from the extracted cell layer and culture medium of human bone cell cultures following incubation in the presence of [35S]sulfate and [3H]leucine. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the synthesized proteoglycans consisted of at least five polydisperse species having median apparent M(r) = 600,000, 400,00, 270,000, 135,000 and 40,000. When chromatographed further on octyl-Sepharose CL-4B, the proteoglycans of the cell layer resolved into three peaks. The unbound fraction (peak cell layer-I) contained a 40,000 species consisting of a single glycosaminoglycan chain with or without peptide. Peak cell layer-II contained three sulfated species on electrophoresis: a 600,000 species uniformly distributed across the peak, a 135,000 species enriched in the ascending limb (similar to bone PG-I as described previously), and a 270,000 species (similar to bone PG-I) enriched in the descending limb. Peak cell layer-III, eluting at 0.2% Triton X-100, was highly enriched in a 400,000 proteoglycan component. When media proteoglycans were chromatographed on octyl-Sepharose, two labeled peaks were found. Peak medium-I (unbound) contained a species exhibiting electrophoretic mobility similar to that of the 400,000 species present in peak cell layer-III. Peak II of the culture medium (medium-II) was apparently identical to that of peak cell layer-II, containing the 600,000, 270,000 and 135,000 species. No appreciable 40,000 species was observed in the medium. Treatment of the 600,000 species with either chondroitinase ABC or ACII generated a core protein preparation with bands of 390,000 and 340,000 on SDS gels. Neither the intact nor the chondroitinase ABC-treated 600,000 species was immunoprecipitated by a purified, polyclonal antiserum raised against the core protein of the large chondroitin sulfate proteoglycan of human articular cartilage. Treatment of the 270,000 and 135,000 proteoglycans with chondroitinase ABC, but not ACII, generated a core protein preparation with bands of 52,000 and 49,000 on SDS gels, indicating that they were dermatan sulfate-containing species. The 400,000 species contained both heparan sulfate and chondroitin sulfate, in approximately a 3:1 labeling ratio. This species changed in electrophoretic mobility following treatment with chondroitinase ABC, heparatinase, or both enzymes in combination, which suggested that it may be a hybrid proteoglycan (i.e. both types of glycosaminoglycan chain on the same core protein). A polyclonal antiserum raised against a mixture of the small proteoglycans purified from the mineral compartment of human bone, immunoprecipitated the intact and chondroitinase ABC-treated 270,000 and 135,000 bone cell proteoglycans. This same antiserum failed to immunoprecipitate either the intact or chondroitinase ABC-treated 600,000 species, indicating that the core proteins prepared from the 270,000 and 135,000 species are antigenically distinct from those of the 600,000 species.

AB - Proteoglycans were isolated by ion-exchange chromatography from the extracted cell layer and culture medium of human bone cell cultures following incubation in the presence of [35S]sulfate and [3H]leucine. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the synthesized proteoglycans consisted of at least five polydisperse species having median apparent M(r) = 600,000, 400,00, 270,000, 135,000 and 40,000. When chromatographed further on octyl-Sepharose CL-4B, the proteoglycans of the cell layer resolved into three peaks. The unbound fraction (peak cell layer-I) contained a 40,000 species consisting of a single glycosaminoglycan chain with or without peptide. Peak cell layer-II contained three sulfated species on electrophoresis: a 600,000 species uniformly distributed across the peak, a 135,000 species enriched in the ascending limb (similar to bone PG-I as described previously), and a 270,000 species (similar to bone PG-I) enriched in the descending limb. Peak cell layer-III, eluting at 0.2% Triton X-100, was highly enriched in a 400,000 proteoglycan component. When media proteoglycans were chromatographed on octyl-Sepharose, two labeled peaks were found. Peak medium-I (unbound) contained a species exhibiting electrophoretic mobility similar to that of the 400,000 species present in peak cell layer-III. Peak II of the culture medium (medium-II) was apparently identical to that of peak cell layer-II, containing the 600,000, 270,000 and 135,000 species. No appreciable 40,000 species was observed in the medium. Treatment of the 600,000 species with either chondroitinase ABC or ACII generated a core protein preparation with bands of 390,000 and 340,000 on SDS gels. Neither the intact nor the chondroitinase ABC-treated 600,000 species was immunoprecipitated by a purified, polyclonal antiserum raised against the core protein of the large chondroitin sulfate proteoglycan of human articular cartilage. Treatment of the 270,000 and 135,000 proteoglycans with chondroitinase ABC, but not ACII, generated a core protein preparation with bands of 52,000 and 49,000 on SDS gels, indicating that they were dermatan sulfate-containing species. The 400,000 species contained both heparan sulfate and chondroitin sulfate, in approximately a 3:1 labeling ratio. This species changed in electrophoretic mobility following treatment with chondroitinase ABC, heparatinase, or both enzymes in combination, which suggested that it may be a hybrid proteoglycan (i.e. both types of glycosaminoglycan chain on the same core protein). A polyclonal antiserum raised against a mixture of the small proteoglycans purified from the mineral compartment of human bone, immunoprecipitated the intact and chondroitinase ABC-treated 270,000 and 135,000 bone cell proteoglycans. This same antiserum failed to immunoprecipitate either the intact or chondroitinase ABC-treated 600,000 species, indicating that the core proteins prepared from the 270,000 and 135,000 species are antigenically distinct from those of the 600,000 species.

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