Rate studies have been employed as a reporter function to probe protein-protein interactions within a biochemically defined reconstituted N-end rule ubiquitin ligation pathway. The concentration dependence for E1-catalyzed HsUbc2b/E2 14kb transthiolation is hyperbolic and yields K m values of 102 ؎ 13 nM and 123 ؎ 19 nM for high affinity binding to rabbit and human E1/Uba1 orthologs. Competitive inhibition by the inactive substrate and product analogs HsUbc2bC88A (K i ‫؍‬ 104 ؎ 15 nM) and HsUbc2bC88S-ubiquitin oxyester (K i ‫؍‬ 169 ؎ 17 nM), respectively, indicates that the ubiquitin moiety contributes little to E1 binding. Under conditions of rate-limiting E3␣-catalyzed conjugation to human ␣-lactalbumin, HsUbc2b-ubiquitin thiolester exhibits a K i of 54 ؎ 18 nM and is competitively inhibited by the substrate analog HsUbc2bC88S-ubiquitin oxyester (K i ‫؍‬ 66 ؎ 29 nM). In contrast, the ligase product analog HsUbc2bC88A exhibits a K i of 440 ؎ 55 nM with respect to the wild type HsUbc2b-ubiquitin thiolester, demonstrating that ubiquitin binding contributes to the ability of E3␣ to discriminate between substrate and product E2. A survey of E1 and E2 isoform distribution in selected cell lines demonstrates that Ubc2 isoforms are the predominant intracellular ubiquitin carrier protein. Intracellular levels of E1 and Ubc2 are micromolar and approximately equal based on in vitro quantitation by stoichiometric 125 I-ubiquitin thiolester formation. Comparison of intracellular E1 and Ubc2 pools with the corresponding ubiquitin pools reveals that most of the free ubiquitin in cells is present as thiolesters to the components of the conjugation pathways. The present data represent the first comprehensive analysis of protein interactions within a ubiquitin ligation pathway.The majority of short-lived cellular proteins are targeted for degradation by the 26 S proteasome in response to assembly of degradation signals on their surface comprising chains of ubiquitin moieties covalently linked through specific lysine residues (1). Target protein specificity for this process is determined in part by a large family of diverse ubiquitin-protein isopeptide ligases (E3) 1 that recognize specific features of the native structure (2-4), transposable trans-acting amino acid sequences (5-7), or exposed regions of non-native conformation (8). The activated ubiquitin required to drive isopeptide bond formation is donated by specific ubiquitin carrier proteins (E2/ Ubc), also termed ubiquitin-conjugating enzymes, in which the ubiquitin carboxyl terminus is bound as a thiolester to a conserved cysteine (9, 10). The apparent specificity of different isopeptide ligases for recognizing a single or limited number of E2 isozymes accounts for the large cohort of related carrier protein isoforms (9 -11). Some E2 moieties may contribute to the substrate specificity of their cognate E3 isozyme because they are able to associate with targets in the absence of ligase (12)(13)(14). In addition, a subset of E2 isozymes catalyzes formation of polyubiquiti...

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Background: Acid α-glucosidase, an enzyme replacement therapy for Pompe disease, is poorly targeted to lysosomes when relying on phosphomannose residues.Results: Fusing IGF-II to acid α-glucosidase resulted in more efficient uptake and glycogen clearance from muscle of Pompe mice.Conclusion: Enhanced binding to the cation-independent mannose 6-phosphate receptor (CI-MPR) enabled improved glycogen clearance in Pompe mice.Significance: BMN 701 is now being tested for Pompe disease in human clinical studies.

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46 kDa cation-dependent MPR (CD-MPR) are key components of the lysosomal enzyme targeting system that bind newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and divert them from the secretory pathway. Previous studies have mapped two high-affinity Man-6-P binding sites of the CI-MPR to domains 1-3 and 9 and one low-affinity site to domain 5 within its 15-domain extracytoplasmic region. A structure-based sequence alignment predicts that domain 5 contains the four conserved residues (Gln, Arg, Glu, Tyr) identified as essential for Man-6-P binding by the CD-MPR and domains 1-3 and 9 of the CI-MPR. Here we show by surface plasmon resonance (SPR) analyses of constructs containing single amino acid substitutions that these conserved residues (Gln-644, Arg-687, Glu-709, Tyr-714) are critical for carbohydrate recognition by domain 5. Furthermore, the N-glycosylation site at position 711 of domain 5, which is predicted to be located near the binding pocket, has no influence on the carbohydrate binding affinity. Endogenous ligands for the MPRs that contain solely phosphomonoesters (Man-6-P) or phosphodiesters (mannose 6-phosphate N-acetylglucosamine ester, Man-P-GlcNAc) were generated by treating the lysosomal enzyme acid alpha-glucosidase (GAA) with recombinant GlcNAc-phosphotransferase and uncovering enzyme (N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase). SPR analyses using these modified GAAs demonstrate that, unlike the CD-MPR or domain 9 of the CI-MPR, domain 5 exhibits a 14-18-fold higher affinity for Man-P-GlcNAc than Man-6-P, implicating this region of the receptor in targeting phosphodiester-containing lysosomal enzymes to the lysosome.

Mannose 6-phosphate (Man-6-P)-dependent trafficking is vital for normal development. The biogenesis of lysosomes, a major cellular site of protein, carbohydrate, and lipid catabolism, depends on the 300-kDa cation-independent Man-6-P receptor (CI-MPR) that transports newly synthesized acid hydrolases from the Golgi. The CI-MPR recognizes lysosomal enzymes bearing the Man-6-P modification, which arises by the addition of GlcNAc-1-phosphate to mannose residues and subsequent removal of GlcNAc by the uncovering enzyme (UCE). The CI-MPR also recognizes lysosomal enzymes that elude UCE maturation and instead display the Man-P-GlcNAc phosphodiester. This ability of the CI-MPR to target phosphodiester-containing enzymes ensures lysosomal delivery when UCE activity is deficient. The extracellular region of the CI-MPR is comprised of 15 repetitive domains and contains three distinct Man-6-P binding sites located in domains 3, 5, and 9, with only domain 5 exhibiting a marked preference for phosphodiester-containing lysosomal enzymes. To determine how the CI-MPR recognizes phosphodiesters, the structure of domain 5 was determined by NMR spectroscopy. Although domain 5 contains only three of the four disulfide bonds found in the other seven domains whose structures have been determined to date, it adopts the same fold consisting of a flattened β-barrel. Structure determination of domain 5 bound to N-acetylglucosaminyl 6-phosphomethylmannoside, along with mutagenesis studies, revealed the residues involved in diester recognition, including Y679. These results show the mechanism by which the CI-MPR recognizes Man-P-GlcNAc-containing ligands and provides new avenues to investigate the role of phosphodiester-containing lysosomal enzymes in the biogenesis of lysosomes.carbohydrate recognition | lectin | lysosome biogenesis | protein targeting L ysosomes are acidified organelles that serve as the terminal degradative compartment of the phagocytic, autophagocytic, and endocytic pathways. The diverse degradative capacity of this organelle depends on a repertoire of over 60 different soluble acid hydrolases (e.g., proteases, glycosidases, nucleases, lipases, and phosphatases) and involves the continual delivery of these newly synthesized hydrolytic enzymes to the lysosome (reviewed in ref.