Folding of intestinal brush border enzymes. Evidence that high-mannose glycosylation is an essential early event.
A polyvalent antiserum which precipitates the native, folded, but not the denatured molecular forms of pig intestinal aminopeptidase N (EC 3.4.11.2) and sucrase-isomaltase (EC 3.2.1.48, EC 3.2.1.10) was used to determine the kinetics of polypeptide folding of the two newly synthesized brush border enzymes. In pulse-labeled mucosal explants, complete synthesis of the polypeptide chains of aminopeptidase N and sucrase-isomaltase required about 2 and 4 min, respectively, whereas maximal antiserum precipitation was acquired with half-times of 4-5 and 8 min, respectively. Fructose, which induces a defective cotranslational high-mannose glycosylation, increased the half-time of polypeptide folding to about 12 min for aminopeptidase N as well as for sucrase-isomaltase. Short-pulse experiments suggested that fructose exerts its effect by slowing the rate of glycosylation, making this partially a posttranslational process. In the presence of fructose, not only the malglycosylated forms but also the electrophoretically normal, high-mannose-glycosylated form of the brush border enzymes were retained in the endoplasmic reticulum and proteolytically degraded. The results obtained demonstrate an intimate interrelationship between glycosylation and polypeptide folding in the synthesis of membrane glycoproteins and, more specifically, indicate that the timing of these two early biosynthetic events is essential for correct polypeptide folding.
Department of Biochemistry C, Panum Institute, University of Copenhagen, Denmark.
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Danielsen
D000626:Aminopeptidases / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D018826:CD13 Antigens; D005632:Fructose / Q000494:pharmacology; D006031:Glycosylation; D006868:Hydrolysis / Q000187:drug effects; D007106:Immune Sera / Q000737:chemistry; D007421:Intestine, Small / Q000737:chemistry / Q000201:enzymology / Q000648:ultrastructure*; D007700:Kinetics; D008358:Mannose / Q000378:metabolism*; D008871:Microvilli / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D009924:Organ Culture Techniques; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry; D011234:Precipitins / Q000737:chemistry; D011487:Protein Conformation; D013394:Sucrase-Isomaltase Complex / Q000737:chemistry* / Q000378:metabolism; D013552:Swine
D007106:Immune Sera; D010455:Peptides; D011234:Precipitins; D005632:Fructose; D013394:Sucrase-Isomaltase Complex; D000626:Aminopeptidases; D018826:CD13 Antigens; D008358:Mannose