Figure 2.

EndoH digestion of recombinant proCLs. Cell lines stably expressing wild type (proCL) or mutants encoding the wild-type glycosylation site plus a novel glycosylation site at Asn-138 (N1N3) or Asn-175 (N2N3). Cells were radiolabeled for 5 h with [³⁵S]methionine, cell lysates were incubated in the presence (+) or absence (−) of endoH to remove high-mannose carbohydrate, and immunoprecipitations were performed with antiserum specific for human proCL. Immunoprecipitated proteins were run on SDS-PAGE and processed for fluorography as described in MATERIALS AND METHODS. Proenzyme (pro), lysosomal single-chain (mature), and two-chain (25) forms of the proteins are indicated. The 5-kDa carboxy-terminal fragment from the two-chain enzyme was run off the gel. The position of the 29-kDa size standard is shown on the left and lane numbers are provided on the bottom.