Journal: The Journal of Biological Chemistry

Article Title: Thrombin Cleavage of Inter-α-inhibitor Heavy Chain 1 Regulates Leukocyte Binding to an Inflammatory Hyaluronan Matrix * ^♦

doi: 10.1074/jbc.M116.755660

Figure Lengend Snippet: Mass spectrometric analysis of thrombin cleavage of bovine HC1. Confluent M-SMCs were treated with poly(I·C) for 18 h at 37 °C. Cells were rinsed three times with PBS and treated with Streptomyces hyaluronidase (100 milliunits/ml) for 10 min to release HC-HA from the cell surface. The supernatants were collected, split into two equal volumes, and incubated without or with thrombin (25 units/ml) for 1 h. Samples were then prepared for mass spectrometry as described under “Experimental Procedures.” A, coverage of all HC1 peptides detected in untreated (blue) or thrombin-treated (red) samples. The two thrombin cleavage sites in bovine HC1 are underlined in red. The N- and C-terminal peptides analyzed in D are in boxes. Tryptic residues are underlined in black; predicted thrombin sites are underlined in red, and a conserved Golgi processing site is underlined in yellow. B, representative Coomassie stain of cell-surface HC-HA supernatants released from M-SMCs by Streptomyces hyaluronidase digestion. Lanes were divided into nine regions for subsequent MS analysis. C, quantification of tryptic HC1 peptides detected in each sample are represented as a percentage of total HC1 peptides excised from indicated regions of B. D, representative chromatogram of a specific N- or C-terminal peptide of HC1 present in both untreated and treated samples. The N-terminal peptide (MAVDAAVDGVVIR) observed in both samples was found only at high mass ranges (data shown for Region 1, blue) of untreated samples but was detected at lower mass ranges (data shown for Region 7, red) of thrombin-treated samples. The C-terminal peptide (ALQMSLAYQFVTPTSMTVR) observed in both samples was detected only high mass ranges of both samples and was not observed at lower mass ranges of thrombin-treated samples. Data are representative of two independent experiments.

Article Snippet: Confluent cultures of M-SMCs were treated with DME/F-12 medium containing 10% FBS with or without poly(I·C) (100 μg/ml) for 18 h. Cultures were rinsed three times with 5 ml of Hanks' BSS and treated with or without thrombin (25 units/ml, 5 ml for a T75-cm 2 culture flask; United States Biochemical Corp.) for 3 h, then rinsed three additional times with Hanks' BSS, and treated with Streptomyces hyaluronidase (100 milliunits/ml, 1 ml/75-cm 2 culture; Seikagaku) for 5 min at 37 °C to release HA-associated material from the cell surface as described previously ( 11 ).

Techniques: Incubation, Mass Spectrometry, Staining

Journal: The Journal of Biological Chemistry

Article Title: Thrombin Cleavage of Inter-α-inhibitor Heavy Chain 1 Regulates Leukocyte Binding to an Inflammatory Hyaluronan Matrix * ^♦

doi: 10.1074/jbc.M116.755660

Figure Lengend Snippet: Thrombin is capable of cleaving HC1 from either cell-surface HC-HA cables or from serum IαI. HA cell-surface layer extracts from untreated, poly(I·C)-treated, or poly(I·C) and thrombin-treated cells were compared by Western blotting. Confluent M-SMCs were treated without or with poly(I·C) for 18 h at 37 °C to form HC-HA cable complexes. Poly(I:C)-treated cells were incubated without or with thrombin (25 units/ml) for 1 h at 37 °C. Cells were rinsed three times with PBS and incubated with Streptomyces hyaluronidase (100 milliunits/ml) for 5 min to release HA-bound cell-surface material. The supernatant was collected and prepared for Western blotting analysis of IαI (A) and HC1 (B). Serum from healthy donors was diluted and incubated without or with thrombin (25 units/ml) for 3 h at 37 °C prior to Western blotting analysis for either IαI (C) or HC1 (D). The green ovals in the schematic models represent HCs attached to bikunin (blue rectangle) via a single CS chain (black line). The schematic with two HCs represents IαI. The schematic with one HC represents PαI. Blots are representative of experiments from three independent patient cell lines and three technical replicates.

Article Snippet: Confluent cultures of M-SMCs were treated with DME/F-12 medium containing 10% FBS with or without poly(I·C) (100 μg/ml) for 18 h. Cultures were rinsed three times with 5 ml of Hanks' BSS and treated with or without thrombin (25 units/ml, 5 ml for a T75-cm 2 culture flask; United States Biochemical Corp.) for 3 h, then rinsed three additional times with Hanks' BSS, and treated with Streptomyces hyaluronidase (100 milliunits/ml, 1 ml/75-cm 2 culture; Seikagaku) for 5 min at 37 °C to release HA-associated material from the cell surface as described previously ( 11 ).

Techniques: Western Blot, Incubation