Characterization of bovine testis mannose 6-phosphate receptors.

Two Man-6-P receptors (MPR-1, 215 kDa and MPR-2, 41-46 kDa) have been isolated from mammalian tissues. The receptors bind ligands carrying covalently bound Man-6-P and are involved in the targeting of acid hydrolases to lysosomes. MPR-1 and MPR-2 were extracted from bovine testis and purified and separated by the sequential use of affinity chromatography and gel filtration. The aggregation state of bovine testis MPR-2 was determined by cross-linking and gel filtration. MPR-2 exists in membranes as a noncovalently-linked dimer and in solution as oligomeric forms, largely as tetramer. The formation of tetramer is affected by the concentration of the receptor in solution. The order of binding of the oligomeric forms to the immobilized ligand agarose-pentamannosephosphate was tetramer $>$ dimer $>$ monomer. MPR-2 contains intrachain disulfide bonds which are of importance in maintaining ligand binding activity and receptor conformation. Bovine testis MPR-2 is comprised of two isoforms (MPR-2A, 45 kDa, and MPR-2B, 41 kDa). Each isoform was purified to near homogeneity by differential centrifugation and affinity chromatography. The isoforms contain a common polypeptide core, but differ in their carbohydrate content. Treatment with specific endoglycosidases demonstrated that each isoform contains two high mannose and/or hybrid and two complex N-linked oligosaccharide chains. The results obtained from treatment of each isoform with endo-$\beta$-galactosidase and neuraminidases and from the use of lectin affinity chromatography suggest that (1) MPR-2A contains a linear polylactosamine sequence(s) with $\sim$5 lactosamine units on one of the complex chains; (2) MPR-2A contains 5-7 sialic acid residues; and (3) MPR-2B lacks polylactosamine sequences and contains only a small amount of sialic acid. Experiments with immobilized lectins revealed that a majority of the outer branches of the complex chains of MPR-2A terminate with sialic acid and those of MPR-2B with $\beta$-galactose. MPR-2A exhibited a lower affinity than MPR-2B for Man-6-P containing ligands. Treatment of MPR-2A with endo-$\beta$-galactosidase and/or neuraminidase followed by affinity chromatography revealed that polylactosamine and sialic acid residues impair the ability of MPR-2A to bind ligands.PhDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105232/1/9116237.pdfDescription of 9116237.pdf : Restricted to UM users only