E. Rodriguez, B.Sc., Dr. P. Roughley, Ph.D.

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General Information Osteoarthritis and Cartilage

Presentation transcript:

Link protein can retard the degradation of hyaluronan in proteoglycan aggregates E. Rodriguez, B.Sc., Dr. P. Roughley, Ph.D. Osteoarthritis and Cartilage Volume 14, Issue 8, Pages 823-829 (August 2006) DOI: 10.1016/j.joca.2006.02.008 Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 1 Scheme of PG aggregate preparation. Aggrecan and LP were isolated from a PG aggregate preparation derived from fetal BEC. Aggrecan was mixed with HA to yield LP-free PG aggregates, and with both HA and LP to yield LP-stabilized PG aggregates. Osteoarthritis and Cartilage 2006 14, 823-829DOI: (10.1016/j.joca.2006.02.008) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 2 Analysis of intact PG aggregates. LP-free and LP-stabilized PG aggregates were analyzed by gel filtration through Sephacryl S-1000 (A). Column fractions were analyzed for GAG content by the DMMB-dye binding assay. The void (Vo) and total (Vt) volumes of the column and the elution position of aggrecan are indicated. The fractions from gel filtration analysis of the LP-stabilized PG aggregate were analyzed for LP content by SDS/PAGE and subsequent immunoblotting for LP (B). The migration position of the two glycosylation variants of intact LP (LP1 and LP2) is indicated. Osteoarthritis and Cartilage 2006 14, 823-829DOI: (10.1016/j.joca.2006.02.008) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 3 Gel filtration analysis of PG aggregates after hyaluronidase treatment. LP-free and LP-stabilized PG aggregates were analyzed by gel filtration through Sephacryl S-1000 following treatment with 0.5units (A) or 0.05 units (B) Streptomyces hyaluronidase per mg PG. Column fractions were analyzed for GAG content by the DMMB-dye binding assay. The void (Vo) and total (Vt) volumes of the column and the elution position of aggrecan are indicated. Osteoarthritis and Cartilage 2006 14, 823-829DOI: (10.1016/j.joca.2006.02.008) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 4 SDS/PAGE analysis of LP after hyaluronidase treatment. The fractions from gel filtration analysis of the LP-stabilized PG aggregate treated with 0.5 units (A) or 0.05 units (B) hyaluronidase (see Fig. 3) were analyzed for LP content by SDS/PAGE and subsequent immunoblotting for LP. The migration position of the two glycosylation variants of intact LP (LP1 and LP2) is indicated. Osteoarthritis and Cartilage 2006 14, 823-829DOI: (10.1016/j.joca.2006.02.008) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 5 Gel filtration analysis of PG aggregates following H2O2 treatment. LP-free and LP-stabilized PG aggregates were analyzed by gel filtration through Sephacryl S-1000 following treatment with 0.3% (A) or 0.03% (B) H2O2. In addition, LP-stabilized PG aggregates were analyzed following treatment with 0.3% H2O2 in the presence of 1μM CuSO4 (C). Column fractions were analyzed for GAG content by the DMMB-dye binding assay. The void (Vo) and total (Vt) volumes of the column and the elution position of aggrecan are indicated. Osteoarthritis and Cartilage 2006 14, 823-829DOI: (10.1016/j.joca.2006.02.008) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 6 SDS/PAGE analysis of LP following H2O2 treatment. LP-stabilized PG aggregate was digested with H2O2 or hyaluronidase, then analyzed for LP content by SDS/PAGE and subsequent immunoblotting for LP. The migration position of the two glycosylation variants of LP (LP1 and LP2) is indicated. Samples are from (1) no treatment, or treatment with (2) 0.03% H2O2, (3) 0.3% H2O2, (4) 0.3% H2O2 plus 1μM CuSO4, (5) treatment with 0.5 units hyaluronidase, and (6) treatment with 0.3% H2O2 plus DETPA. Osteoarthritis and Cartilage 2006 14, 823-829DOI: (10.1016/j.joca.2006.02.008) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 7 Diagmatic representation of LP function. PG aggregates are depicted as central cores of HA interacting with multiple aggrecan molecules that are packed maximally. LP-free aggregates are depicted with spaces between aggrecan molecules that allow ready access for hyaluronidases or free radicals. LP-stabilized aggregates are depicted as possessing a continuous protein coat along the HA which limits hyaluronidase or free radical action. In reality, LP-stabilized aggregates may possess a lower degree of aggrecan/LP substitution and would possess an incomplete protein coat and have an increased susceptibility to hyaluronidase or radical attack. Osteoarthritis and Cartilage 2006 14, 823-829DOI: (10.1016/j.joca.2006.02.008) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions