Isolation and characterization of a cDNA that encodes the core protein of the cytolytic granule proteoglycan in rat natural killer cells

Abstract

In this report, we describe the transcription of messenger ribonucleic acid (mRNA) specific for the core protein of chondroitin sulfate proteoglycan (CSPG), the entire sequence of the message for CSPG core protein and the presence of CSPG in the granules of a highly purified population of recombinant interleukin-2 (rIL-2)-stimulated rat natural killer (NK), i.e. adherent lymphokine-activated Killer (A-LAK), cells. The presence of CSPG in A-LAK cell granules was demonstrated by a variety of biochemical and immunologic methods. Further, we have demonstrated the presence of a 1.1-kilobase (kb) transcript for the core protein of CSPG by Northern blot analysis using a specific probe (pPG6) derived from a rat yolk sac tumor cell line (L-2). Three cell types that contained CSPG in granules produced a transcript of 1.1 kb, whereas L-2 cells, which localize CSPG on the cell surface, produced a transcript of 1.3 kb. A complementary DNA (cDNA) library was prepared from rat A-LAK cells and the gene for the CSPG core protein was cloned. From approximately 1.2 X 10(5) recombinant phages, 5 positive clones were obtained. The longest clone, PG-NK-5, was sequenced in its entirety and it was found to encode the entire sequence of the CSPG core protein. The other 4 clones were partially sequenced and were identical to PG-NK-5. Comparison of the sequence of PG-NK-5 with pPG6 indicated that they were nearly identical. However, all NK-cell-derived cDNAs contained a poly(A) tail that started 20 basepairs upstream from other published sequences for CSPG core proteins. These data represent the first description of the sequence of the core protein of CSPG contained in NK cells.