FIGURE 5.

The pathological heavy chain modification of HA is present in lung tissue from a patient with acute severe asthma. Lung tissue from an individual with acute severe asthma was minced in a normalized volume of PBS (per wet weight of tissue) and incubated with or without Streptomyces hyaluronidase, an HA14 oligomer as a heavy chain acceptor, and TSG-6 to transfer heavy chains (panel A). The proteins were electrophoretically separated, transferred, and stained with an antibody against IαI (green). Molecular weight standards are shown in red. Lane 1 shows untreated lung tissue. The HC-HA complex was too large to enter the gel, but when digested with hyaluronidase (HAase) (lane 2) the heavy chains were released and migrated as a single band at ∼83 kDa. In lane 3, an HA oligo, 14 monosaccharides in length, was added to the minced tissue to detect endogenous TSG-6 activity. As a positive control, exogenous recombinant TSG-6 was also added with the HA14 (lane 4). Panel B shows four replicates of healthy control (Ctrl) lung tissue treated with and without hyaluronidase (similar to the asthmatic tissue in lanes 1 and 2 of panel A). The green lobules in the schematic models represent HCs attached to bikunin (blue rectangle) via a single chondroitin sulfate chain (black line). The schematic with two HCs represents IαI. The schematic with one HC represents pre-IαI. The asthmatic tissue in panel A was from a rare, large surgical biopsy of a single patient. Surgical biopsies of asthmatics are not a recommended diagnostic procedure as asthma is determined by other clinical criteria. For this reason we are unable to present more replicates from the asthmatic cohort.