title: Rat brain contains high levels of mannose-6-phosphorylated glycoproteins including lysosomal enzymes and palmitoyl-protein thioesterase, an enzyme implicated in infantile neuronal lipofuscinosis.
abstract: Mannose 6-phosphate (Man-6-P) is a posttranslational carbohydrate modification typical of newly synthesized acid hydrolases that signals targeting from the Golgi apparatus to the lysosome via Man-6-P receptors (MPRs). Using iodinated cation independent MPR as a probe in a Western blot assay, we surveyed levels of Man-6-P glycoproteins in a number of different rat tissues. Considerable variation was observed with respect to total amounts and types of Man-6-P glycoproteins in the different tissues. Brain contained 2-8-fold more Man-6-P glycoproteins than other tissues, with relative abundance being brain >> testis approximately heart > lung approximately kidney approximately ovary approximately spleen > skeletal muscle approximately liver approximately serum. Analysis of 16 different lysosomal enzyme activities revealed that brain contains lower activities than other tissues which suggested that decreased removal of Man-6-P results in increased levels of Man-6-P glycoproteins. This was directly demonstrated by comparing activities of phosphorylated lysosomal enzymes, purified by immobilized MPR affinity chromatography, with total activities. The phosphorylated forms accounted for a considerable proportion of the MPR-targeted activities measured in brain (on average, 36.2%) but very little in lung, kidney, and liver (on average, 5.5, 2.3, and 0. 7%, respectively). Man-6-P glycoproteins were also isolated from rat brain by MPR affinity chromatography on a preparative scale. Of the 18 bands resolvable by SDS-polyacrylamide gel electrophoresis, seven bands were NH2-terminally sequenced and identified as the known lysosomal enzymes cathepsin L, cathepsin A, cathepsin D, alpha-galactosidase A, arylsulfatase A, and alpha-iduronidase. One of the major Man-6-P glycoproteins was identified as palmitoyl protein thioesterase, which was not previously thought to be lysosomal. This finding raises important questions about the cellular location and function of palmitoyl protein thioesterase, mutations in which result in the neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis.
authors.affiliation: Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854, USA.
authors.forename: D E
authors.initials: DE
authors.lastname: Sleat
authors.forename: I
authors.initials: I
authors.lastname: Sohar
authors.forename: H
authors.initials: H
authors.lastname: Lackland
authors.forename: J
authors.initials: J
authors.lastname: Majercak
authors.forename: P
authors.initials: P
authors.lastname: Lobel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology* / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D008360:Mannosephosphates / Q000378:metabolism*; D008969:Molecular Sequence Data; D009472:Neuronal Ceroid-Lipofuscinoses / Q000201:enzymology*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013869:Thiolester Hydrolases / Q000378:metabolism*
chemical_list: D006023:Glycoproteins; D008360:Mannosephosphates; C027693:mannose-6-phosphate; D013869:Thiolester Hydrolases; C095299:palmitoyl-protein thioesterase