Structure of the human GlcNAc-1-phosphotransferase αβ subunits reveals regulatory mechanism for lysosomal enzyme glycan phosphorylation.

Vertebrates use the mannose 6-phosphate (M6P)-recognition system to deliver lysosomal hydrolases to lysosomes. Key to this pathway is N-acetylglucosamine (GlcNAc)-1-phosphotransferase (PTase) that selectively adds GlcNAc-phosphate (P) to mannose residues of hydrolases. Human PTase is an αβγ heterohexamer with a catalytic core and several peripheral domains that recognize and bind substrates.

Species-specific differences in the processing of acid α-glucosidase are due to the amino acid identity at position 201.

Acid α-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes glycogen to glucose. Deficiency of GAA causes Pompe disease. Mammalian GAA is synthesized as a precursor of ~110,000 Da that is N-glycosylated and targeted to the lysosome via the M6P receptors.

Structural basis for recognition of phosphodiester-containing lysosomal enzymes by the cation-independent mannose 6-phosphate receptor.

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).

Performance evaluation of the new ADVIA Centaur system cyclosporine assay (single-step extraction).

Background: Cyclosporine (CsA) monitoring is essential for transplant success. We report a performance study of the recently released, fully automated Siemens ADVIA Centaur CsA assay.

Methods: Whole blood samples from 248 transplant patients were prepared using a new 1-step extraction method.

Functions of the alpha, beta, and gamma subunits of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase.

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an alpha(2)beta(2)gamma(2) hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Using a multifaceted approach, including analysis of acid hydrolase phosphorylation in mice and fibroblasts lacking the gamma subunit along with kinetic studies of recombinant alpha(2)beta(2)gamma(2) and alpha(2)beta(2) forms of the transferase, we have explored the function of the alpha/beta and gamma subunits. The findings demonstrate that the alpha/beta subunits recognize the protein determinant of acid hydrolases in addition to mediating the catalytic function of the transferase.

Cation-independent mannose 6-phosphate receptor: a composite of distinct phosphomannosyl binding sites.

The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR), which contains multiple mannose 6-phosphate (Man-6-P) binding sites that map to domains 3, 5, and 9 within its 15-domain extracytoplasmic region, functions as an efficient carrier of Man-6-P-containing lysosomal enzymes. To determine the types of phosphorylated N-glycans recognized by each of the three carbohydrate binding sites of the CI-MPR, a phosphorylated glycan microarray was probed with truncated forms of the CI-MPR. Surface plasmon resonance analyses using lysosomal enzymes with defined N-glycans were performed to evaluate whether multiple domains are needed to form a stable, high affinity carbohydrate binding pocket.

Glycoengineered acid alpha-glucosidase with improved efficacy at correcting the metabolic aberrations and motor function deficits in a mouse model of Pompe disease.

Improving the delivery of therapeutics to disease-affected tissues can increase their efficacy and safety. Here, we show that chemical conjugation of a synthetic oligosaccharide harboring mannose 6-phosphate (M6P) residues onto recombinant human acid alpha-glucosidase (rhGAA) via oxime chemistry significantly improved its affinity for the cation-independent mannose 6-phosphate receptor (CI-MPR) and subsequent uptake by muscle cells. Administration of the carbohydrate-remodeled enzyme (oxime-neo-rhGAA) into Pompe mice resulted in an approximately fivefold higher clearance of lysosomal glycogen in muscles when compared to the unmodified counterpart.

Reduction in facial photodamage by a topical growth factor product.

A topical gel containing a proprietary mixture of over 110 growth factors, cytokines, and soluble matrix proteins secreted by human dermal fibroblasts was evaluated for safety and efficacy in the treatment of mild to severe facial photodamage. In a double-blind study, 60 subjects were randomly assigned to receive either active gel or the vehicle and applied twice daily for 6 months along with a moisturizing cleanser and sunscreen. Efficacy (profilometry, photography, and clinical assessment) and safety (adverse event reporting) measures were evaluated at 0, 3, and 6 months.

Domain 5 of the cation-independent mannose 6-phosphate receptor preferentially binds phosphodiesters (mannose 6-phosphate N-acetylglucosamine ester).

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.

Structural requirements for efficient processing and activation of recombinant human UDP-N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase.

Mannose 6-phosphate-modified N-glycans are the determinant for intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. The enzyme responsible for the initial step in the synthesis of mannose 6-phosphate is UDP-N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosmine-1-phosphotransferase(GlcNAc-phosphotransferase). GlcNAc-phosphotransferase is a multisubunit enzyme with an alpha2beta2gamma2 arrangement that requires a detergent for solubilization.

Mucolipidosis II (I-cell disease) and mucolipidosis IIIA (classical pseudo-hurler polydystrophy) are caused by mutations in the GlcNAc-phosphotransferase alpha / beta -subunits precursor gene.

Mucolipidosis II (MLII; I-cell disease) and mucolipidosis IIIA (MLIIIA; classical pseudo-Hurler polydystrophy) are diseases in which the activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent or reduced, respectively. In the absence of mannose phosphorylation, trafficking of lysosomal hydrolases to the lysosome is impaired. In these diseases, mistargeted lysosomal hydrolases are secreted into the blood, resulting in lysosomal deficiency of many hydrolases and a storage-disease phenotype.

The alpha- and beta-subunits of the human UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase [corrected] are encoded by a single cDNA.

Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an alpha2beta2gamma2-subunit structure was proposed.

Hyaluronan biosynthesis by class I streptococcal hyaluronan synthases occurs at the reducing end.

Previous studies reached different conclusions about whether class I hyaluronan synthases (HASs) elongate hyaluronic acid (HA) by addition to the reducing or the nonreducing end. Here we used two strategies to determine the direction of HA synthesis by purified class I HASs from Streptococcus equisimilis and Streptococcus pyogenes. In the first strategy we used each of the two UDP-sugar substrates separately to pulse label either the beginning or the end of HA chains.

Lysosomal acid alpha-glucosidase consists of four different peptides processed from a single chain precursor.

Pompe's disease is caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). GAA is synthesized as a 110-kDa precursor containing N-linked carbohydrates modified with mannose 6-phosphate groups. Following trafficking to the lysosome, presumably via the mannose 6-phosphate receptor, the 110-kDa precursor undergoes a series of complex proteolytic and N-glycan processing events, yielding major species of 76 and 70 kDa.

Automated flow cytometric analysis of blood cells in cerebrospinal fluid: analytic performance.

We compared the performance of an automated method for obtaining RBC and WBC counts and WBC differential counts in cerebrospinal fluid (CSF) samples with the reference manual method. Results from 325 samples from 10 worldwide clinical sites were used to demonstrate the accuracy, precision, and linearity of the method. Accuracy statistics for absolute cell counts showed a high correlation between methods, with correlation coefficients for all reportable absolute counts greater than 0.

Human mannose 6-phosphate-uncovering enzyme is synthesized as a proenzyme that is activated by the endoprotease furin.

N-Acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, also known as "uncovering" enzyme (UCE), is localized in the trans-Golgi network, where it removes a covering N-acetylglucosamine from the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Here we show that UCE is synthesized as an inactive proenzyme that is activated by the endoprotease furin, which cleaves an RARLPR/D sequence to release a 24-amino acid propiece. As furin is localized in the trans-Golgi network, newly synthesized UCE is inactive until it reaches this terminal Golgi compartment.

Cloning and expression of human core 1 beta1,3-galactosyltransferase.

The common core 1 O-glycan structure Galbeta1--> 3GalNAc-R is the precursor for many extended mucin-type O-glycan structures in animal cell surface and secreted glycoproteins. Core 1 is synthesized by the transfer of Gal from UDP-Gal to GalNAcalpha1-R by core 1 beta3-galactosyltransferase (core 1 beta3-Gal-T). Amino acid sequences from purified rat core 1 beta3-Gal-T (Ju, T.

Purification, characterization, and subunit structure of rat core 1 Beta1,3-galactosyltransferase.

The O-linked oligosaccharides (O-glycans) in mammalian glycoproteins are classified according to their core structures. Among the most common is the core 1 disaccharide structure consisting of Galbeta1-->3GalNAcalpha1-->Ser/Thr, which is also the precursor for many extended O-glycan structures. The key enzyme for biosynthesis of core 1 O-glycan from the precursor GalNAc-alpha-Ser/Thr is UDP-Gal:GalNAc-alpha-Ser/Thr beta3-galactosyltransferase (core1 beta3-Gal-T).