Characterization of the human gene that encodes the peptide core of secretory granule proteoglycans in promyelocytic leukemia HL-60 cells and analysis of the translated product.
Based upon the deduced amino acid sequence of a cDNA (cDNA-H4) that had been proposed to encode the peptide core of an eosinophil and a HL-60 cell secretory granule proteoglycan, a 16-amino acid peptide was synthesized. This peptide was then used to elicit rabbit antibodies for study of the translation and post-translational modification of this gene product in hematopoietic cells. When HL-60 cells were radiolabeled for 2 min with [35S]methionine, a protein that migrated in a sodium dodecyl sulfate-polyacrylamide electrophoresis gel with a Mr of 20,000 was immunoprecipitated with the IgG fraction of the anti-peptide serum. Kinetic experiments revealed that within 10 min this radiolabeled precursor protein was converted in HL-60 cells into an Mr approximately 150,000 chondroitin sulfate proteoglycan intermediate. After a 20-min to 1-h chase, this [35S]methionine- or [35S]sulfate-labeled proteoglycan intermediate lost its antigenicity, presumably due to proteolysis of its N terminus. A human genomic library was probed under conditions of high stringency with cDNA-H4 to isolate genomic clones that contain the gene that encodes this proteoglycan peptide core. This gene spans approximately 15 kilobases and consists of three exons. The first exon encodes the 5'-untranslated region of the mRNA transcript, as well as the entire 27-amino acid signal peptide of the translated molecule. The second exon encodes a 49-amino acid region of the peptide core, predicted to be the N terminus of the molecule after its proteolytic processing in the endoplasmic reticulum. The third exon encodes the remainder of the molecule, including its glycosaminoglycan attachment, serine-glycine repeat region. As assessed by S1 nuclease mapping and primer extension analysis, the transcription-initiation site in HL-60 cells for this gene resides 53 base pairs upstream from the translation-initiation site.
Nicodemus
C F
CF
Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Avraham
S
S
Austen
K F
KF
Purdy
S
S
Jablonski
J
J
Stevens
R L
RL
D000595:Amino Acid Sequence; D001483:Base Sequence; D005091:Exons; D006801:Humans; D007163:Immunosorbent Techniques; D007438:Introns; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism*; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis; D011509:Proteoglycans / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D013431:Sulfates / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D046911:Macromolecular Substances; D011509:Proteoglycans; D013431:Sulfates; C055916:secretory granule proteoglycan; D008715:Methionine