Plasma membrane arabinogalactan proteins

Sammanfattning: Arabinogalactan proteins (AGPs) are proteoglycans found at the outer side of the plasma membrane and in the extracellular matrix of plants. To be able to study the plasma membrane bound AGPs, a method using SDS-agarose gel electrophoresis (SDS-AGE) was developed to overcome the problems associated with electrophoretic separation of highly glycosylated proteins. AGPs were partially purified by Triton X-114 fractionation of plasma membranes isolated by aqueous polymer two-phase partitioning of a microsomal fraction from sugar beet leaves. By the use of isoelectric focusing and SDS-AGE, in combination with monoclonal antibodies specific for AGPs, it was shown that sugar beet plasma membrane AGPs consist of several isoforms with the apparent molecular weights 82 and 97 kDa. The isoforms migrate as two well separated bands in the SDS-AGE system. 1D and 2D protein gel blots, using the SDS-AGE method, probed with monoclonal antibodies (MAbs) proved to be very useful for the analysis of the plasma membrane AGPs and were used throughout the investigation. If sugar beet leaves were excised and incubated at room temperature prior to homogenization at 0°C, additional AGP bands appeared with the apparent molecular weights 120, 170 and 210 kDa. In the presence of hydrogen peroxide and horseradish peroxidase, purified AGPs could be cross-linked in vitro to form the 170 kDa AGP band. As compared to younger leaves, older leaves contained increased amounts of primarily the 170 and 120 kDa AGPs. The sugar beet plasma membrane AGPs were studied with a set of MAbs recognizing different AGP carbohydrate epitopes. Some of these epitopes were shown to be expressed on only one or a few of the AGPs, and some of the AGP epitopes are under developmental control. Our results suggest a mechanism for generating new epitopes at the plasma membrane by hydrogen peroxide dependent oxidative cross-linking. When plasma membrane AGPs were studied in a number of closely and distantly related species, the presence of certain epitopes and the size of the AGPs were shown to vary with species. In suspension cultures of carrot cells, one of the AGP specific epitopes can be used to track embryogenic potential. This epitope is also present on a molecule that affects meristematic activity in Arabidopsis roots.