An assay to detect glycoproteins that contain mannose 6-phosphate.
We have developed a broadly applicable solid phase assay for quantitative and qualitative analysis of phosphorylated lysosomal enzymes and other glycoproteins that contain mannose 6-phosphate (Man6-P) residues. Samples are immobilized on membranes and proteins that contain Man6-P are detected using an iodinated, soluble form of the cation-independent Man6-P receptor. The assay is highly specific, sensitive, and linear over 3 orders of magnitude. The technique has two general applications. First, it can be used in a dot blot format to analyze large numbers of samples for total levels of Man6-P glycoproteins. Second, it can be used in a western blot format to identify Man6-P glycoproteins that have been fractionated by polyacrylamide gel electrophoresis. Specific applications of this novel assay include structural characterization of glycoproteins, diagnosis of mucolipidosis II and III, and screening of pharmaceuticals for lysosomotropic activity. Other potential applications are discussed.
Valenzano
K J
KJ
Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey, Piscataway 08854.
Kallay
L M
LM
Lobel
P
P
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry*; D006801:Humans; D015151:Immunoblotting; D008360:Mannosephosphates / Q000032:analysis* / Q000378:metabolism; D008567:Membranes, Artificial; D008722:Methods; D051379:Mice; D011145:Polyvinyls; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors; D016707:Tissue Fixation; D005465:Fluorocarbon Polymers
D006023:Glycoproteins; D008360:Mannosephosphates; D008567:Membranes, Artificial; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C027693:mannose-6-phosphate