title: Polymorphism of prolactin secreted by human prolactinoma cells: immunological, receptor binding, and biological properties of the glycosylated and nonglycosylated forms.
abstract: Multiple forms of PRL differing in their physicochemical and biological characteristics have been described. We have analyzed the molecular forms of human (h) PRL released in culture by pure hPRL-secreting tumors with a particular attention to glycosylated hPRL. The prolactinoma cells from six different tumors released, in serum-free conditions, 10-28 mg hPRL. The combination of polyacrylamide gel electrophoresis and immunoblotting techniques using a [125I]anti-hPRL monoclonal antibody allowed qualitative and quantitative analysis of the hPRL variants. The ratio of the glycosylated 25,000-mol wt form (G-hPRL) to the 23,000-mol wt nonglycosylated monomeric hPRL (NG-hPRL) varied from 0.13 to 0.25. Under the conditions of our studies, cleaved forms of the hormone (19,000 and 15,000 mol wt) accounted for less than 5% of the total immunoreactivity. G- and NG-hPRL were subsequently purified by gel filtration and lectin affinity chromatography. G-hPRL appeared fully sensitive to endoglycosidase F digestion, further supporting the presence of a freely accessible N-linked carbohydrate chain. When assayed for their ability to react with polyclonal antibodies directed against hPRL in a competitive RIA, G-hPRL was 3 times less immunoreactive than NG-hPRL. However, both types of hPRL exhibited superimposable displacement curves when tested in an immunoassay using an anti-hPRL monoclonal antibody. In binding studies using crude rabbit mammary gland membranes G-hPRL was half as potent as NG-hPRL. In stimulating the growth of the Nb2 lymphoma cell line, G-hPRL was 50% less active than NG-hPRL. Thus 1) under basal conditions, hPRL undergoes partial and variable glycosylation; 2) glycosylation of the hormone may modulate its immunoreactivity; 3) glycosylation of hPRL not only lowers its mammary gland receptor binding capacity but also its growth-promoting activity.
mesh_terms: D000236:Adenoma / Q000378:metabolism*; D000818:Animals; D002455:Cell Division; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007118:Immunoassay; D008297:Male; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D051379:Mice; D008970:Molecular Weight; D010911:Pituitary Neoplasms / Q000378:metabolism*; D011110:Polymorphism, Genetic; D011388:Prolactin / Q000235:genetics / Q000378:metabolism*; D011863:Radioimmunoassay; D011869:Radioligand Assay; D011981:Receptors, Prolactin / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
chemical_list: D011981:Receptors, Prolactin; D011388:Prolactin; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase