Biosynthesis, processing, and targeting of sphingolipid activator protein (SAP )precursor in cultured human fibroblasts. Mannose 6-phosphate receptor-independent endocytosis of SAP precursor

Sphingolipid activator proteins (SAPs) are essential cofactors for the lysosomal degradation of glycosphingolipids with short oligosaccharide chains by acidic exohydrolases. SAP-A, -B, -C, and -D derive from proteolysis of a 73-kDa glycoprotein, the SAP precursor. In the present publication, we studied the intracellular transport and the endocytosis of SAP precursor in human skin fibroblasts. Our data indicate that SAP precursor bears phosphate residues on noncomplex carbohydrate chains linked to the SAP-C and the SAP-D domain and sulfate residues on complex carbohydrate chains located within the SAP-A, -C, and possibly the SAP-D domain. Treatment of fibroblasts with either bafilomycin A1 or 3-methyladenine indicates that proteolytic cleavage of SAP precursor begins as early as in the late endosomes. To determine whether targeting of SAP precursor depends on mannose 6-phosphate residues, we analyzed the processing of SAP precursor in I-cell disease fibroblasts. In these cells nearly normal amounts of newly synthesized SAP-C were found, although secretion of SAP precursor was enhanced 2-3-fold. Moreover, SAP-C could be localized to lysosomal structures by indirect immunofluorescence in normal and in I-cell disease fibroblasts. Mannose 6-phosphate was not found to interfere significantly with endocytosis of SAP precursor. Normal fibroblasts internalized SAP precursor secreted from I-cells nearly as efficiently as the protein secreted from normal cells. To our surprise, deglycosylated SAP precursor was taken up by mannose 6-phosphate receptor double knock out mouse fibroblasts more efficiently than the glycosylated protein. We propose that intracellular targeting of SAP precursor to lysosomes is only partially dependent on mannose 6-phosphate residues, whereas its endocytosis occurs in a carbohydrate-independent manner.     

Re-expression of the mannose 6-phosphate receptors in receptor-deficient fibroblasts. Complementary function of the two mannose 6-phosphate receptors in lysosomal enzyme targeting

We have previously generated primary embryonic fibroblasts lacking either the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (MPR) or the cation-dependent MPR, two trans-membrane proteins that bind the mannose 6-phosphate (Man-6-P) recognition marker on soluble lysosomal enzymes (Ludwig, T., Munier-Lehmann, H., Bauer, U., Hollinshead, M., Ovitt, C., Lobel, P., and Hoflack, B.(1994) EMBO J. 13, 3430-3437). These two cell types partially missort phosphorylated lysosomal enzymes. Using two-dimensional gel electrophoresis, we show here that they secrete, in a large part, different phosphorylated ligands. In order to better understand the sorting function of the MPRs, we have re-expressed each MPR in MPR-negative fibroblasts. We show that the MPRs have similar capacities for transporting the bulk of the newly synthesized lysosomal enzymes and that they target individual ligands with various efficiencies. However, high levels of one MPR do not fully compensate for the absence of the other, demonstrating that the two MPRs have complementary targeting functions, perhaps by recognizing different features on lysosomal enzymes. The analysis of the phosphorylated oligosaccharides shows that the ligands missorted in the absence of the cation-dependent MPR are slightly but significantly depleted in oligosaccharides with two Man-6-P residues, when compared with those missorted in the absence of the cation-independent MPR. While these results could explain some differences between the structure and the sorting function of the two MPRs, they strongly suggest that the reason why cells express two different but related MPRs is to maintain an efficient Man-6-P-dependent targeting process that could be potentially regulated by MPR expression.     

Functional characterization of the N-glycosylation sites of human acid sphingomyelinase by site-directed mutagenesis

Most soluble lysosomal enzymes require a mannose-6-phosphate recognition marker present on asparagine-linked oligosaccharides for proper targeting to lysosomes. We have determined the influence of the six potential N-linked oligosaccharide chains of human acid sphingomyelinase (ASM) on catalytic activity, targeting, and processing of the enzyme. Each N-glycosylation site was modified by site-directed mutagenesis and subsequently expressed in COS-1 cells. Evidence is presented that five of these sites are used. Elimination of the four N-terminal glycosylation sites does not disturb lysosomal targeting, processing, or enzymatic activity. However, removal of the two C-terminal N-glycosylation sites inhibits the formation of mature enzyme. Absence of glycosylation site five resulted in rapid cleavage of the primary translation product to an enzymatically inactive protein which accumulated inside the endoplasmic reticulum/Golgi, whereas deletion of glycosylation site six led to the formation of an inactive ASM precursor, also retained inside the endoplasmic reticulum/Golgi. Our results also provide evidence that the site of early proteolytic cleavage of newly synthesized ASM must be located between the second and third glycosylation sites.     

Lysine-based structure responsible for selective mannose phosphorylation of cathepsin D and cathepsin L defines a common structural motif for lysosomal enzyme targeting

Previous studies have shown that lysine residues on the surface of cathepsins and other lysosomal proteins are a shared component of the recognition structure involved in mannose phosphorylation. In this study, the involvement of specific lysine residues in mannose phosphorylation of cathepsin D was explored by site-directed mutagenesis. Mutation of two lysine residues in the mature portion of the protein, Lys-203 and Lys-293, cooperated to inhibit mannose phosphorylation by 70%. Other positively charged residues could not substitute for lysine at these positions, and comparison of thermal denaturation curves for the wild type and mutant proteins indicated that the inhibition could not be explained by alterations in protein folding. Structural comparisons of the two lysine residues with those required for phosphorylation of cathepsin L, using models generated from recently acquired crystal structures, revealed several relevant similarities. On both molecules, the lysine residues were positioned approximately 34 A apart (34.06 A for cathepsin D and 33.80 A for cathepsin L). When the lysine pairs were superimposed, N-linked glycosylation sites on the two proteins were found to be oriented so that oligosaccharides extending out from the sites could share a common region of space. Further similarities in the local environments of the critical lysines were also observed. These results provide details for a common lysosomal targeting structure based on a specific arrangement of lysine residues with respect to each other and to glycosylation sites on the surface of lysosomal proteins.     

Phosphorylated oligosaccharides in lysosomal enzymes: identification of alpha-N-acetylglucosamine(1)phospho(6)mannose diester groups

In human fibroblasts, the recognition of lysosomal enzymes by cell surface receptors is mediated by mannose 6-phosphate residues located on oligosaccharides that can be cleaved by endo-beta-N-acetylglucosaminidase H. About half of these oligosaccharides, as isolated from beta-hexosaminidase and cathepsin D secreted by human skin fibroblasts, are anionic. Most of these are resistant to alkaline phosphatase. The resistance is due to alpha-N-acetylglucosamine residues linked to mannose 6-phosphate by a phosphodiester bond. The major phosphorylated oligosaccharides contain one and two and possibly three phosphate groups blocked by N-acetylglucosamine. Besides the blocked phosphate groups these oligosaccharides contain a common inner core consisting of Man alpha 1,6-(Man alpha 1,3)Man alpha 1,6(Man alpha 1,3)Man beta GlcNAc and either one or two alpha 1,2-linked mannose residues.     

Cathepsin D associates with lysosomal membranous protein

The membrane-association of early biosynthetic form of cathepsin D has been demonstrated in hepatoma cells, and this membrane-association is not mediated by mannose 6-phosphate residues, implying that a mannose 6-phosphate receptor-independent mechanism operates in the sorting of cathepsin D. In this paper, to demonstrate whether cathepsin D is associated with the lysosomal membranes, an in vitro binding experiment was carried out employing lysosomal cathepsin D or microsomal procathepsin D isolated from rat liver. Immunoblotting analysis revealed that an intermediate form of cathepsin D was associated with the lysosomal membranes; this lysosomal membrane-associated cathepsin D was released from the membranes by washing with Na2CO3 (pH 10.6) but not with solutions containing mannose 6-phosphate. This suggested that cathepsin D associates with the membranes by ionic-interaction, and that the membrane-associated cathepsin D resides as a peripheral membrane protein in the lysosomal membrane fraction. To confirm that the intermediate form of cathepsin D specifically interacts with the lysosomal integral membrane proteins, the lysosomal membrane fraction was treated with trypsin and the binding experiment was conducted. The result showed that the binding capacity of cathepsin D to the lysosomal membranes was apparently abolished and cathepsin D did not rebind to the membranes. These data suggest that the intermediate form of cathepsin D is preferentially recognized by the lysosomal membranous protein which complements the mannose 6-phosphate receptor-dependent intracellular sorting mechanism.     

Oligosaccharide binding characteristics of the molecular chaperones calnexin and calreticulin

Calnexin and calreticulin are homologous molecular chaperones of the endoplasmic reticulum. Their binding to newly synthesized glycoproteins is mediated, at least in part, by a lectin site that recognizes the early N-linked oligosaccharide processing intermediate, Glc1Man9GlcNAc2. We compared the oligosaccharide binding specificities of calnexin and calreticulin in an effort to determine the basis for reported differences in their association with various glycoproteins. Using mono-, di-, and oligosaccharides to inhibit the binding of Glc1Man9GlcNAc2 to calreticulin and to a truncated, soluble form of calnexin, we show that the entire Glc alpha 1-3Man alpha 1-2Man alpha 1-2Man structure, extending from the alpha 1-3 branch point of the oligosaccharide core, is recognized by both proteins. Furthermore, analysis of the binding of monoglucosylated oligosaccharides containing progressively fewer mannose residues suggests that for both proteins the alpha 1-6 mannose branch point of the oligosaccharide core is also essential for recognition. Consistent with their essentially identical substrate specificities, calnexin and calreticulin exhibited the same relative affinities when competing for binding to the Glc1Man9GlcNAc2 oligosaccharide. Thus, differential glycoprotein binding cannot be attributed to differences in the lectin specificities or binding affinities of calnexin and calreticulin. We also examined the effects of ATP, calcium, and disulfide reduction on the lectin properties of calnexin and calreticulin. Whereas oligosaccharide binding was only slightly enhanced for both proteins in the presence of high concentrations of a number of adenosine nucleotides, removal of bound calcium abrogated oligosaccharide binding, an effect that was largely reversible upon readdition of calcium. Disulfide reduction had no effect on oligosaccharide binding by calnexin, but binding by calreticulin was inhibited by 70%. Finally, deletion mutagenesis of calnexin and calreticulin identified a central proline-rich region characterized by two tandem repeat motifs as a segment capable of binding oligosaccharide. This segment bears no sequence homology to the carbohydrate recognition domains of other lectins.     

Purification and characterization of human lymphoblast N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase

The enzyme N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase (EC 3.1.4.45; uncovering enzyme) catalyzed the removal of N-acetylglucosamine from the N-acetylglucosamine-alpha-phospho-mannose portion of selected lysosomal enzyme oligosaccharide chains, thereby forming the mannose 6-phosphate signal which is responsible for the targeting of these lysosomal enzymes for transport into lysosomes. The uncovering enzyme has been purified approximately 7000-fold to electrophoretic homogeneity from Epstein-Barr virus-transformed human lymphoblast cells. The purification sequence involves solubilizing this membrane-bound enzyme with Tergitol NP-10, affinity chromatography on Lentil lectin-Sepharose 4B, ion-exchange chromatography on DEAE-Sephacel, chromatography on zinc(II)-IDA-Sepharose 6B, and preparative SDS-PAGE electrophoresis. The purified enzyme migrated as a single band of 114 kDa which was coincident with enzyme activity on analytical SDS-PAGE electrophoresis. Characterization studies of the purified enzyme demonstrated that catalytic activity was maximal at pH 6.95 and that the enzyme retained full activity following incubation for 10 min at 60 degrees C. No requirement was found for a divalent cation, but Zn2+, Hg2+, and Cu2+ were found to reduce the enzyme's activity by 30-40%. The highest catalytic efficiency was observed with N-acetylglucosamine-phospho-methylmannoside as a substrate while uridine diphosphate-N-acetylglucosamine, N-acetylglucosamine-phosphomannose-uteroferrin, and N-acetylglucosamine-phosphate were also cleaved by the enzyme with decreasing efficiency. Acetamino-deoxycastanospermine was a potent inhibitor of the human enzyme with a Ki of 0.35 microM, while N-acetylglucosamine phosphate (Ki 1.58 mM) and N-acetylglucosamine (Ki 5.1 mM) inhibited the enzyme to a lesser degree.     

Activation and oligomerization of aspartylglucosaminidase

Secretory, membrane, and lysosomal proteins undergo covalent modifications and acquire their secondary and tertiary structure in the lumen of the endoplasmic reticulum (ER). In order to pass the ER quality control system and become transported to their final destinations, many of them are also assembled into oligomers. We have recently determined the three-dimensional structure of lysosomal aspartylglucosaminidase (AGA), which belongs to a newly discovered family of homologous amidohydrolases, the N-terminal nucleophile hydrolases. Members of this protein family are activated from an inactive precursor molecule by an autocatalytic proteolytic processing event whose exact mechanism has not been thoroughly determined. Here we have characterized in more detail the initial events in the ER required for the formation of active AGA enzyme using transient expression of polypeptides carrying targeted amino acid substitutions. We show that His124 at an interface between two heterodimers of AGA is crucial for the thermodynamically stable oligomeric structure of AGA. Furthermore, the side chain of Thr206 is essential both for the proteolytic activation and enzymatic activity of AGA. Finally, the proper geometry of the residues His204-Asp205 seems to be crucial for the activation of AGA precursor polypeptides. We propose here a reaction mechanism for the activation of AGA which could be valid for homologous enzymes as well.     

Prolonged dilation improves an unsatisfactory primary result of femoropopliteal artery angioplasty: usefulness of a perfusion balloon catheter

D-[U-14C]Glucosamine was rapidly taken up by oat coleoptile segments and metabolized to radioactive UDP-N-acetylglucosamine, which acted as specific glycosyl donor for the synthesis of glycolipids and cytosolic, membrane-bound and cell-wall glycoproteins. Cell-wall glycoproteins were solubilized from the walls by either cell-wall-degrading enzymes or chemical extractants. The solubilized cell-wall glycoproteins in the presence of peptide N-glycosidase F released oligosaccharide chains higher than seven glycosidic residues. The combined action of peptide N-glycosidase F and N-acetyl-beta-D-glucosaminidase on cell-wall glycoproteins indicated the presence of N-acetylglucosamine residues beta-1,2-linked to mannose. Less than 9% of the radioactive oligosaccharide chains was released from the solubilized cell-wall glycoproteins when treated with 0.5 M NaOH at 20 degrees, whereas more than 45% of the radioactivity was released in the presence of 1 M NaOH at 50 degrees. The high hydrolytic sensitivity of cell-wall glycoproteins to peptide N-glycosidase F, N-acetyl-beta-D-glucosaminidase and NaOH at 50 degrees indicated that most N-acetylglucosamine residues were incorporated into N-linked cell-wall glycoproteins. Further evidence of this was obtained by the use of inhibitors of biosynthesis and processing of N-linked glycoproteins.     

Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal neurodegenerative disease whose defective gene has remained elusive. A molecular basis for LINCL was determined with an approach applicable to other lysosomal storage diseases. When the mannose 6-phosphate modification of newly synthesized lysosomal enzymes was used as an affinity marker, a single protein was identified that is absent in LINCL. Sequence comparisons suggest that this protein is a pepstatin-insensitive lysosomal peptidase, and a corresponding enzymatic activity was deficient in LINCL autopsy specimens. Mutations in the gene encoding this protein were identified in LINCL patients but not in normal controls.     

Relative contributions of subcutaneous and intermuscular fat to yields and predictability of retail product, fat trim, and bone in beef carcasses

Previous studies have suggested that the phosphoenolpyruvate:mannose phosphotransferase system of Streptococcus salivarius consists of a nonphosphorylated enzyme II domain that functions in tandem with a separate enzymatic complex called III(Man). The III(Man) complex is believed to be composed of two protein dimers with molecular masses of approximately 72 kDa. Analysis of these proteins by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate has indicated that one dimer is composed of two 38.9-kDa subunits called IIIH(Man), and the other of two 35.2-kDa subunits called IIIL(Man). This study was undertaken to determine (1) the number and nature of the phosphorylated residue(s) on IIIH(Man) and IIIL(Man) and the phosphorylation sequence allowing the transfer of the phosphoryl group from HPr(His approximately P) to the mannose:PTS substrates; (2) whether IIIH(Man) and IIIL(Man) originate from two different genes or result from a posttranslational modification; and (3) whether these two proteins are involved in the phosphorylation of 2-deoxyglucose, a substrate of the phosphoenolpyruvate:mannose phosphotransferase system. We showed that both IIIH(Man) and IIIL(Man) were phosphorylated on two histidine residues. One phosphate bond was heat-labile (phosphorylation at the N1 position of the imidazole ring), while the second was heat-resistant (phosphorylation at the N3 position of the imidazole ring). The sequence of the first phosphorylation site was deduced by comparing the N-terminal amino acid sequence of both forms of III(Man) with IIA domains of the EII-mannose family. The sequences of both forms were identical over the 15 first amino acids, that is, MIGIIIASHGKFAEG. The sequence of the second phosphorylation site was determined for IIIL(Man) as IHGQVATNxTP. Hence, IIIH(Man) and IIIL(Man) are PTS proteins of the IIAB type and should be renamed IIABH(Man) and IIABL(Man). IIABH(Man) and IIABL(Man) had different peptide profiles after digestion with proteases, indicating that these two proteins are encoded by two different genes. In vitro PEP-dependent phosphorylation assays conducted with a spontaneous mutant devoid of both forms of IIAB(Man) suggested that the phosphoenolpyruvate:mannose phosphotransferase system of S. salivarius is composed of an uncharacterized nonphosphorylated membrane component that works in tandem with IIABL(Man). The physiological functions of IIABH(Man) remain unknown.     

Release and analysis of polypeptides and glycopolypeptides from formalin-fixed, paraffin wax-embedded tissue

Archival tissue specimens are commonly stored as formalin-fixed, paraffin wax-embedded blocks. Formalin fixation facilitates excellent morphological preservation, and the immunoreactivity of many antigens is preserved, but formalin-induced chemical cross-linking of proteins renders them insoluble and inaccessible to standard biochemical extraction and analytical methods. Thus, biochemical analysis of tissue components identified by histochemistry, with the advantage of long-term clinical follow-up, is precluded. We have applied cyanogen bromide cleavage, a technique used routinely for fragmenting proteins for sequencing experiments, to solubilize transferrin polypeptides and glycopolypeptides from formalin-fixed, paraffin wax-embedded rat liver. Cyanogen bromide cleaves protein specifically at methionine residues, yielding a predictable array of polypeptide fragments. Subsequent oligosaccharide analysis of the transferrin glycopolypeptides by anion exchange chromatography confirmed that, in addition to successful release of polypeptide chains, sialylated oligosaccharide structures remained intact after cyanogen bromide cleavage. This approach may have wide applicability to a range of research interests in which correlation of tissue biochemistry with long-term follow-up is advantageous.     

Targeting of Yersinia Yop proteins into the cytosol of HeLa cells: one-step translocation of YopE across bacterial and eukaryotic membranes is dependent on SycE chaperone

Pathogenic Yersiniae adhere to and kill macrophages by targeting some of their Yop proteins into the eukaryotic cytosol. There is debate about whether YopE targeting proceeds as a direct translocation of polypeptide between cells or in two distinct steps, each requiring specific signals for YopE secretion across the bacterial envelope and for translocation into the eukaryotic cytosol. Here, we used the selective solubilization of the eukaryotic plasma membrane with digitonin to measure Yop targeting during Yersinia infections of HeLa cells. YopE, YopH, YopM and YopN were found in the eukaryotic cytosol but not in the extracellular medium. When bound to SycE chaperone in the Yersinia cytoplasm, YopE residues 1-100 are necessary and sufficient for the targeting of hybrid neomycin phosphotransferase. Electron microscopic analysis failed to detect an extracellular intermediate of YopE targeting, suggesting a one-step translocation mechanism.     

Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP)

Sphingolipid activator proteins SAP-A, -B, -C and -D (also called saposins) are generated by proteolytic processing from a 73 kDa precursor and function as obligatory activators of lysosomal enzymes involved in glycosphingolipid metabolism. Although the SAP precursor can be recognized by the mannose-6-phosphate (M-6-P) receptor and shuttled directly from the secretory pathway to the lysosome, a substantial fraction of newly synthesized precursor is secreted from the cell where it may participate in sphingolipid transport and signaling events. Re-uptake of the secreted precursor is mediated by high-affinity cell surface receptors that are apparently distinct from the M-6-P receptor. We found that the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic receptor that is expressed on most cells, can mediate cellular uptake and lysosomal delivery of SAP precursor. Additional in vivo experiments in mice revealed that the mannose receptor system on macrophages also participates in precursor internalization. We conclude that SAP precursor gains entry into cells by at least three independent receptor mechanisms including the M-6-P receptor, the mannose receptor and LRP.     

Overview of N- and O-linked oligosaccharide structures found in various yeast species

Yeast and most higher eukaryotes utilize an evolutionarily conserved N-linked oligosaccharide biosynthetic pathway that involves the formation of a Glc3Man9GlcNAc2-PP-dolichol lipid-linked precursor, the glycan portion of which is co-translationally transferred in the endoplasmic reticulum (ER) to suitable Asn residues on nascent polypeptides. Subsequently, ER processing glycohydrolases remove the three glucoses and, with the exception of Schizosaccharomyces pombe, a single, specific mannose residue. Processing sugar transferases in the Golgi lead to the formation of core-sized structures (Hex<15GlcNac2) as well as cores with an extended poly-alpha1,6-Man 'backbone' that is derivatized with various carbohydrate side chains in a species-specific manner (Hex50-200GlnNAc2). In some cases these are short alpha1,2-linked Man chains with (Saccharomyces cerevisiae) or without (Pichia pastoris) alpha1,3-Man caps, while in other yeast (S. pombe), the side chains are alpha1,2-linked Gal, some of which are capped with beta-1,3-linked pyruvylated Gal residues. Charged groups are also found in S. cerevisiae and P. pastoris N-glycans in the form of mannose phosphate diesters. Some pathogenic yeast (Candida albicans) add poly-beta1,2-Man extension through a phosphate diester to their N-glycans, which appears involved in virulence. O-Linked glycan synthesis in yeast, unlike in animal cells where it is initiated in the Golgi using nucleotide sugars, begins in the ER by addition of a single mannose from Man-P-dolichol to selected Ser/Thr residues in newly made proteins. Once transported to the Golgi, sugar transferases add one (C. albicans) or more (P. pastoris) alpha1,2-linked mannose that may be capped with one or two alpha1,3-linked mannoses (S. cerevisiae). S. pombe is somewhat unique in that it synthesizes a family of mixed O-glycans with additional alpha1,2-linked Man and alpha1,2- and 1, 3-linked Gal residues.     

The two mannose 6-phosphate binding sites of the insulin-like growth factor-II/mannose 6-phosphate receptor display different ligand binding properties

The two mannose 6-phosphate (Man-6-P) binding sites of the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) have been localized to domains 1-3 and 7-9, and studies have shown that Arg435 in domain 3 and Arg 1334 in domain 9 are essential for Man-6-P binding. To determine whether the IGF-II/MPR containing a single Man-6-P binding site is functional, clonal mouse L cell lines stably transfected with either mutant bovine IGF-II/MPR cDNA, containing substitutions at position 435 and/or 1334, or the wild type receptor cDNA were assayed for their ability to sort lysosomal enzymes to the lysosome. Mutant receptors containing a single Man-6-P binding site were approximately 50% less efficient than the wild type receptor in the overall targeting of lysosomal enzymes to the lysosome. Mutant receptors containing a substitution at Arg1334 (Dom9(Ala)), in contrast to those containing a substitution at Arg435 (Dom3(Ala)), were unable to target cathepsin D and beta-hexosaminidase to the lysosome. Equilibrium binding assays using 125I-labeled beta-glucuronidase demonstrated that Dom3(Ala) and Dom9(Ala) had a Kd of 2.0 and 4.3 nM, respectively. In addition, Dom3(Ala), unlike Dom9(Ala), was unable to completely dissociate from ligand under acidic pH conditions. These data indicate that the two Man-6-P binding sites of the IGF-II/MPR are not functionally equivalent.     

High-performance liquid chromatography of proteins

Incubation of liver microsomes with GDP [14C] mannose leads to the formation of lipid-linked derivatives of [14C] mannose, a dolichol phosphate monosaccharide and dolichol pyrophosphate oligosaccharides. Standard procedures for separating these two types of compounds from each other were found to be deficient in that fractions thought to contain only dolichol pyrophosphate oligosaccharides are contaminated with dolichol phosphate mannose. This paper presents a column chromatographic procedure which conveniently separates the products of an 8 min labeling experiment into two components; dolichol phosphate [14C]mannose and a [14C]-mannose containing oligosaccharide which is also lipid bound. When this oligosaccharide is released from the lipid by hydrolysis and chromatographed on Sephadex G-50 or G-15 it gives a single peak with an indicated molecular weight of 1100. However, when this released oligosaccharide is chromatographed on concanavalin A Sepharose it is resolved into two peaks suggesting that there may be 2 oligosaccharide of approximately the same size but different structures. After brief periods of labeling with GDP [14C]mannose (5 s) an additional oligosaccharide of 3 to 4 sugar residues can be found in the dolichol pyrophosphate oligosaccharides fraction. Incubation of liver microsomes with UDP [14C]glucose or UDP[14C]galactose produces oligosaccharide components containing 7--8 sugar residues. Labeling of microsomes with UDP[14C]acetylglucosamine gives rise to three different components, including a lipid bound oligosaccharide containing 3- 5 sugar residues.     

Plasma proteins glycosylation and its alteration in disease

Most plasma proteins are glycoproteins, that is, they possess oligosaccharide chains attached to the polypeptide core. These oligosaccharides have important structural and functional roles; they serve as recognition markers (ligands), especially for lectin receptors, thus modulating the glycoprotein interactions. Protein glycosylation is a posttranslational event which depends on the proteic core and biosynthetic cell type and results in a set of microheterogeneous forms (glycoforms) of an individual glycoprotein. Under pathological conditions an alteration of the glycosylation pattern of plasma glycoproteins occurs. So, degalactosylated IgG and IgA1 detected in rheumatoid arthritis and IgA nephropathy, respectively, are implicated in the pathogenic mechanisms. Alteration of transferrin, alpha 1-acid glycoprotein and alpha-fetoprotein glycosylation (reduced sialylation and increased branching of oligosaccharide chains) occurs in liver diseases. In inflammations and infections the alteration is dependent on the disease studied, while increased sialylation and fucosylation of acute-phase proteins are detected in cancer sera. Lectin-based methods have been developed for clinical purposes, in order to improve the diagnosis, prognosis evaluation, or treatment monitoring.     

Profiles of published social work practitioners: who wrote and why

Pasteurella multocida was examined for glucose and mannose transport. P. multocida was shown to possess a phosphoenolpyruvate (PEP):mannose phosphotransferase system (PTS) that transports glucose as well as mannose and was functionally similar to the Escherichia coli mannose PTS. Phosphorylated proteins with molecular masses similar to those of E. coli mannose PTS proteins were visualized when incubated with 32P-PEP. The presence of an enzyme IIAGlc which could play an important role in regulation, as described in other Gram-negative bacteria, was detected. The enzymes of the pentose-phosphate pathway were present in P. multocida growth on glucose. The activity of 6-phosphofructokinase (the key enzyme of the Embden-Meyerhof pathway (EMP)), was very low in cell extracts, suggesting that EMP is not the major pathway for glucose catabolism.