Recombinant human epidermal growth factor precursor is a glycosylated membrane protein with biological activity.
NIH 3T3 cells were transfected with cDNA corresponding to human kidney prepro-epidermal growth factor (preproEGF) under control of the inducible mouse metallothionein promoter. The synthesis of recombinant human EGF precursor by these cells has provided us with a model system for analysis of the structure and activity of this precursor. In transfected cells, the precursor was present as an intrinsic 170-kilodalton membrane protein as well as a soluble protein in the extracellular medium; both forms were N glycosylated. Glycosylation of the EGF precursor was determined by (i) the direct incorporation of [3H]mannose and [3H]glucosamine, (ii) metabolic labeling in the presence or absence of glycosylation inhibitors, (iii) enzymatic cleavage of the precursor by N-glycanase or endoglycosidase II, and (iv) lectin chromatography. Recombinant human preproEGF was purified by affinity chromatography, using wheat germ lectin and antibodies to human EGF. The intact precursor was biologically active. Purified preparations of preproEGF (i) competed with 125I-labeled EGF for binding to the EGF receptor in intact fibroblast cells, (ii) activated the intrinsic tyrosine kinase activity of the EGF receptor in membrane preparations, and (iii) sustained the growth of a mouse keratinocyte cell line that is dependent on EGF for growth. These results suggest that proteolytic processing of the precursor may not be essential for its biological function.
Mroczkowski
B
B
Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232.
Reich
M
M
Chen
K
K
Bell
G I
GI
Cohen
S
S
D001667:Binding, Competitive; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism; D006026:Glycoside Hydrolases; D006031:Glycosylation; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D008565:Membrane Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; C042652:epidermal growth factor precursor; D004815:Epidermal Growth Factor; D004247:DNA; D066246:ErbB Receptors; D006026:Glycoside Hydrolases; D005944:Glucosamine; D008358:Mannose