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1.
The correct intracellular sorting of lysosomal enzymes such as arylsulfatase A depends on the presence of mannose 6-phosphate residues on high mannose type oligosaccharides. The arylsulfatase A cDNA contains three potential N-glycosylation sites, two of which are utilized. We have mutated one or two of the N-glycosylation sites and analyzed the glycosylation, phosphorylation, and intracellular sorting of the mutant arylsulfatase A polypeptides. The results show that each of the three glycosylation sites (I, II, and III) can be glycosylated, but glycosylation at sites I and II is mutually exclusive. In mutants with one oligosaccharide side chain at positions I, II, or III all side chains can acquire mannose 6-phosphate residues irrespective of their location. This demonstrates spatial flexibility of the phosphotransferase, which specifically recognizes lysosomal enzymes and initiates the addition of mannose 6-phosphate residues on oligosaccharide side chains. However, these mutants have different intracellular sorting efficiencies and seem to use different (mannose 6-phosphate receptor-dependent and -independent) sorting pathways.  相似文献   

2.
Based on the published bovine DNase sequence (Liao, T.-H., Salnikow, J., Moore, S., and Stein, W. H. (1973) J. Biol. Chem. 248, 1489-1495), the ovine DNase sequence is derived from the amino acid compositions of isolated short peptides covering all regions of the intact polypeptide. The sequence is substantiated by results of automated Edman degradation of the intact polypeptide and of the two middle CNBr fragments, and by elucidation of the complete sequence of the COOH-terminal CNBr peptide. The 12 changes from bovine to ovine DNase are at residues 22 (Ala to Ser), 29 (Val to Leu), 35 (Val to Ala), 54 (Tyr to Asp), 62 (Thr to Ser), 83 (Leu to Val), 121 (His to Pro), 127 (Glu to Ala), 132 (Ala to Pro), 159 (His to Asp), 163 (Val to Ile), and 231 (Ala to Val). A minor genetic variant form of ovine DNase has Val at residue 163. The data from automated Edman degradation of the largest CNBr peptide of bovine DNase show that the published bovine DNase sequence is in error and that an Ile-Val-Arg tripeptide must be inserted between Arg-27 and Arg-28. The corrected sequence is substantiated by two peptides covering this region each with three amino acids more than the published sequence. Comparison of the bovine, ovine, and porcine DNase sequences reveals the following: with the revised bovine sequence, all three DNase sequences can be aligned without a gap; all three DNases have a carbohydrate side chain at Asn-18, but only porcine DNase has carbohydrate at Asn-106; there are 12 changes between bovine and ovine DNases, 56 between bovine and porcine, and 50 between ovine and porcine; there are six highly variable regions and four invariable ones; bovine and ovine DNases have the same length while porcine DNase is longer by 2 amino acid residues at the COOH terminus; the residues around the nucleotide-binding site, the four pairs of salt bridges, and the essential His-134 groups are not changed.  相似文献   

3.
Mannose for mammalian glycan biosynthesis can be imported directly from the medium, derived from glucose or salvaged from endogenous or external glycans. All pathways must generate mannose 6-phosphate, the activated form of mannose. Imported or salvaged mannose is directly phosphorylated by hexokinase, whereas fructose 6-phosphate from glucose is converted to mannose 6-phosphate by phosphomannose isomerase (PMI). Normally, PMI provides the majority of mannose for glycan synthesis. To assess the contribution of PMI-independent pathways, we used PMI-null fibroblasts to study N-glycosylation of DNase I, a highly sensitive indicator protein. In PMI-null cells, imported mannose and salvaged mannose make a significant contribution to N-glycosylation. When these cells were grown in mannose-free medium along with the mannosidase inhibitor, swainsonine, to block the salvage pathways, N-glycosylation of DNase I was almost completely eliminated. Adding approximately 13 microm mannose to the medium completely restored normal glycosylation. Treatment with bafilomycin A(1), an inhibitor of lysosomal acidification, also markedly reduced N-glycosylation of DNase I, but in this case only 8 microm mannose was required to restore full glycosylation, indicating that a nonlysosomal source of mannose made a significant contribution. Glycosylation levels were greatly also reduced in glycoconjugate-free medium, when endosomal membrane trafficking was blocked by expression of a mutant SKD1. From these data, we conclude that PMI-null cells can salvage mannose from both endogenous and external glycoconjugates via lysosomal and nonlysosomal degradation pathways.  相似文献   

4.
The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.  相似文献   

5.
The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.  相似文献   

6.
The determinants on the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) required for lysosomal enzyme sorting have been analyzed. Mouse L cells deficient in the mannose 6-phosphate/insulin-like growth factor-II receptor were transfected with normal bovine CD-MPR cDNA or cDNAs containing mutations in the 67-amino acid cytoplasmic tail and assayed for their ability to target the lysosomal enzyme cathepsin D to lysosomes. Cells expressing the wild-type bovine CD-MPR sorted 67 +/- 2% of newly synthesized cathepsin D compared with the base-line value of 47 +/- 1%. The presence of mannose 6-phosphate in the medium did not affect the efficiency of cathepsin D sorting, indicating that the routing of the ligand-receptor complex is completely intracellular. Mutant receptors with the carboxyl-terminal His-Leu-Leu-Pro-Met67 residues deleted or replaced with alanines sorted cathepsin D below the base-line value. A mutant receptor with the outermost Pro-Met residues replaced with alanines sorted cathepsin D better than the wild-type receptor, indicating that the essential residues for sorting are the His-Leu-Leu sequence. Disruption of a putative casein kinase II phosphorylation site at Ser57 had no detectable effect on sorting. The mutant receptor with the five-amino acid deletion was able to bind to a phosphopentamannose affinity column, proving that its ligand binding site was grossly intact. Resialylation experiments showed that this mutant receptor recycled from the cell surface to the Golgi at a rate similar to the normal CD-MPR, indicating that the defect in sorting is at the level of the Golgi.  相似文献   

7.
Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site-directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion-coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gln 9, Arg 41, Tyr 76, Arg 111, Asn 170, Tyr 175, and Tyr 211 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I-like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I-like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn2+-dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions.  相似文献   

8.
Mannose phosphorylation of N-linked oligosaccharides by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is a key step in the targeting of lysosomal enzymes in mammalian cells and tissues. The selectivity of this process is determined by lysine-based phosphorylation signals shared by lysosomal enzymes of diverse structure and function. By introducing new glycosylation sites at several locations on the surface of mouse procathepsin L and modeling oligosaccharide conformations for sites that are phosphorylated, it was shown that the inherent flexibility of N-linked oligosaccharides can account for the specificity of the transferase for oligosaccharides at different locations on the protein. By using this approach, the physical relationship between the lysine-based signal and the site of phosphorylation of mannose residues was determined. The analysis also revealed the existence of additional independent lysine-based phosphorylation signals on procathepsin L, which account for the low level of phosphorylation observed when the primary Lys-54/Lys-99 signal is ablated. Mutagenesis of residues that surround Lys-54 and Lys-99 and demonstration of mannose phosphorylation of a glycosylated derivative of green fluorescent protein provide strong evidence that the cathepsin L phosphorylation signal is a simple structure composed of as few as two well placed lysine residues.  相似文献   

9.
You YO  van der Donk WA 《Biochemistry》2007,46(20):5991-6000
Lantibiotic synthetases catalyze the dehydration of Ser and Thr residues in their peptide substrates to dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively, followed by the conjugate addition of Cys residues to the Dha and Dhb residues to generate the thioether cross-links lanthionine and methyllanthionine, respectively. In this study ten conserved residues were mutated in the dehydratase domain of the best characterized family member, lacticin 481 synthetase (LctM). Mutation of His244 and Tyr408 did not affect dehydration activity with the LctA substrate whereas mutation of Asn247, Glu261, and Glu446 considerably slowed down dehydration and resulted in incomplete conversion. Mutation of Lys159 slowed down both steps of the net dehydration: phosphorylation of Ser/Thr residues and the subsequent phosphate elimination step to form the dehydro amino acids. Mutation of Arg399 to Met or Leu resulted in mutants that had phosphorylation activity but displayed greatly decreased phosphate elimination activity. The Arg399Lys mutant retained both activities, however. Similarly, the Thr405Ala mutant phosphorylated the LctA substrate but had compromised elimination activity. Finally, mutation of Asp242 or Asp259 to Asn led to mutant enzymes that lacked detectable dehydration activity. Whereas the Asp242Asn mutant retained phosphate elimination activity, the Asp259Asn mutant was not able to eliminate phosphate from a phosphorylated substrate peptide. A model is presented that accounts for the observed phenotypes of these mutant enzymes.  相似文献   

10.
The structures of the native fructose-1,6-bisphosphatase (Fru-1,6-Pase), from pig kidney cortex, and its fructose 2,6-bisphosphate (Fru-2,6-P2) complexes have been refined to 2.8 A resolution to R-factors of 0.194 and 0.188, respectively. The root-mean-square deviations from the standard geometry are 0.021 A and 0.016 A for the bond length, and 4.4 degrees and 3.8 degrees for the bond angle. Four sites for Fru-2,6-P2 binding per tetramer have been identified by difference Fourier techniques. The Fru-2,6-P2 site has the shape of an oval cave about 10 A deep, and with other dimensions about 18 A by 12 A. The two Fru-2,6-P2 binding caves of the dimer in the crystallographically asymmetric unit sit next to one another and open in opposite directions. These two binding sites mutually exchange their Arg243 side-chains, indicating the potential for communication between the two sites. The beta, D-fructose 2,6-bisphosphate has been built into the density and refined well. The oxygen atoms of the 6-phosphate group of Fru-2,6-P2 interact with Arg243 from the adjacent monomer and the residues of Lys274, Asn212, Tyr264, Tyr215 and Tyr244 in the same monomer. The sugar ring primarily contacts with the backbone atoms from Gly246 to Met248, as well as the side-chain atoms, Asp121, Glu280 and Lys274. The 2-phosphate group interacts with the side-chain atoms of Ser124 and Lys274. A negatively charged pocket near the 2-phosphate group includes Asp118, Asp121 and Glu280, as well as Glu97 and Glu98. The 2-phosphate group showed a disordered binding perhaps because of the disturbance from the negatively charged pocket. In addition, Asn125 and Lys269 are located within a 5 A radius of Fru-2,6-P2. We argue that Fru-2,6-P2 binds to the active site of the enzyme on the basis of the following observations: (1) the structure similarity between Fru-2,6-P2 and the substrate; (2) sequence conservation of the residues directly interacting with Fru-2,6-P2 or located at the negatively charged pocket; (3) a divalent metal site next to the 2-phosphate group of Fru-2,6-P2; and (4) identification of some active site residues in our structure, e.g. tyrosine and Lys274, consistent with the results of the ultraviolet spectra and the chemical modification. The structures are described in detail including interactions of interchain surfaces, and the chemically modifiable residues are discussed on the basis of the refined structures.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

11.
The Hsp70 and Hsp40 chaperone machine plays critical roles in protein folding, membrane translocation, and protein degradation by binding and releasing protein substrates in a process that utilizes ATP. The activities of the Hsp70 family of chaperones are recruited and stimulated by the J domains of Hsp40 chaperones. However, structural information on the Hsp40–Hsp70 complex is lacking, and the molecular details of this interaction are yet to be elucidated. Here we used steered molecular dynamics (SMD) simulations to investigate the molecular interactions that occur during the dissociation of the auxilin J domain from the Hsc70 nucleotide-binding domain (NBD). The changes in energy observed during the SMD simulation suggest that electrostatic interactions are the dominant type of interaction. Additionally, we found that Hsp70 mainly interacts with auxilin through the surface residues Tyr866, Arg867, and Lys868 of helix II, His874, Asp876, Lys877, Thr879, and Gln881 of the HPD loop, and Phe891, Asn895, Asp896, and Asn903 of helix III. The conservative residues Tyr866, Arg867, Lys868, His874, Asp876, Lys877, and Phe891 were also found in a previous study to be indispensable to the catalytic activity of the DnaJ J domain and the binding of it with the NBD of DnaK. The in silico identification of the importance of auxilin residues Asn895, Asp896, and Asn903 agrees with previous mutagenesis and NMR data suggesting that helix III of the J domain of the T antigen interacts with Hsp70. Furthermore, our data indicate that Thr879 and Gln881 from the HPD loop are also important as they mediate the interaction between the bovine auxilin J domain and Hsc70.  相似文献   

12.
The structures of the predominant high mannose oligosaccharides present in a human IgM myeloma protein (Patient Wa) have been determined. The IgM glycopeptides, produced by pronase digestion, were fractionated on DEAE-cellulonalysis shows that glycopeptide I contains Asn, Pro, Ala, Thr, and His and glycopeptide II contains Asn, Val, and Ser, which are the same amino acids found in the sequences around Asn 402 and Asn 563 respectively, to which high mannose oligosaccharides are attached in IgM (Patient Ou) (Putnman, F.W., Florent, G., Paul, C., Shinoda, T., and Shimizu, A. (1973) Science 182, 287-290). The high mannose glycopeptides in IgM (Wa) exhibit heterogeneity in the oligosaccharide portion. Structural analysis of the major oligosaccharides indicates that the simplest structure is: (see article of journal). The larger oligosaccharides present have additional mannose residues linked alpha 1 yields 2 to terminal mannose residues in the above structure. Glycopeptide I contains primarily Man5 and Man6 species, while glycopeptide II contains Man6 and Man8 species. The two Man6 oligosaccharides have different branching patterns.  相似文献   

13.
Theil R  Scheit KH 《The EMBO journal》1983,2(7):1159-1163
Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH2-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.  相似文献   

14.
T Watanabe  N Wada  J Y Chou 《Biochemistry》1992,31(12):3051-3058
Human germ cell alkaline phosphatase (GCAP), which shares 98% amino acid sequence identity with the placental AP (PLAP), is expressed by malignant trophoblasts. Protein sequence analysis suggests that the Ser residue at position 92 is the putative active site of GCAP which contains two recognition sequences (Asn122-Thr-Thr124 and Asn249-Arg-Thr251) for asparagine-linked glycosylation. To examine the roles of the Ser residue and glycan moieties on GCAP activity and processing, we altered the GCAP cDNA by site-directed mutagenesis and expressed the GCAP mutants in COS-1 cells. Substitution of Ser-92 with either a Thr (S92T) or an Ala (S92A) residue yielded a GCAP devoid of catalytic activity, suggesting that the Ser codon 92 is the active site of GCAP. Six GCAP mutants that lack one or both glycosylation sites were constructed by substituting either Asn-122 or Asn-249 with an Asp residue or either Thr-124 or Thr-251 with an Ala residue. The mature GCAP migrated as a 65-kDa product, but GCAP mutants lacking one or both glycosylation sites migrated as 62- or 58-kDa polypeptides, respectively, indicating that both sites were glycosylated. All six glycosylated mutants were active enzymatically and, in addition, were equally sensitive to heat, L-leucine, and EDTA inhibition as the parental enzyme. GCAP as well as its two active-site and six glycosylation mutants could be released from the plasma membrane of transfected COS-1 cells by the proteinase bromelain.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

15.
The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.  相似文献   

16.
Xu D  Ballou DP  Massey V 《Biochemistry》2001,40(41):12369-12378
Three residues in the active site of the flavoprotein phenol hydroxylase (PHHY) were independently changed by site-directed mutagenesis. One of the mutant forms of PHHY, Tyr289Phe, is reduced by NADPH much slower than is the wild-type enzyme, although it has a slightly higher redox potential than the wild-type enzyme. In the structure of the wild-type enzyme, residue Tyr289 is hydrogen-bonded with the FAD when the latter is at the "out" position but has no direct contact with the flavin when it is "in". The oxidative half-reaction of PHHY is not significantly affected by this mutation, contrary to the concept that Tyr289 is a critical residue in the hydroxylation reaction [Enroth, C., Neujahr, H., Schneider, G., and Lindqvist, Y. (1998) Structure 6, 605-617; Ridder, L., Mullholland, A. J., Rietjens, I. M. C. M., and Vervoort, J. (2000) J. Am. Chem. Soc. 122, 8728-8738]. Tyr289 may help stabilize the FAD in the out conformation where it can be reduced by NADPH. For the Asp54Asn mutant form of PHHY, the initial step of the oxidative half-reaction is significantly slower than for the wild-type enzyme. Asp54Asn utilizes less than 20% of the reduced flavin for hydroxylating the substrate with the remainder forming H(2)O(2). Similar changes are observed when Arg281, a residue between Asp54 and the solvent, is mutated to Met. These two residues are suggested to be part of the active site environment the enzyme provides for the flavin cofactor to function optimally in the oxidative half-reaction. In the construction of the mutant forms of PHHY, it was determined that 11 of the previously reported amino acid residues in the sequence of PHHY were incorrect.  相似文献   

17.
We recently reported the cDNA cloning, sequence, and expression of the human cation-independent mannose 6-phosphate receptor (hCI-MPR) (Oshima, A., Nolan, C. M., Kyle, J. W., Grubb, J. H., and Sly, W. S. (1988) J. Biol. Chem. 263, 2553-2562). The sequence of the hCI-MPR was virtually identical to that of the human insulin-like growth factor II receptor cDNA (Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). To test the role of the putative bifunctional receptor in intracellular sorting of acid hydrolases, we studied its effect on lysosomal enzyme transport following gene transfer to receptor-negative cells. Receptor-negative mouse P388D1 cells were transfected with a cDNA construct containing the entire coding sequence of hCI-MPR under the control of the mouse metallothionine I promoter. Stable transformants were isolated and characterized. The expressed hCI-MPR was localized in membranes including the plasma membrane, bound mannose 6-phosphate containing ligands, and mediated endocytosis which could be specifically blocked by mannose 6-phosphate. We next measured the effect of the expressed hCI-MPR on intracellular and secreted acid hydrolases. The intracellular activity of the lysosomal marker enzymes beta-glucuronidase and beta-hexosaminidase increased up to 2-fold following transformation. In addition, expression of the receptor greatly reduced the fraction of acid hydrolases secreted. These phenotypic changes in the transformed cell lines support the proposed role of the cation-independent mannose 6-phosphate receptor in intracellular sorting and targeting of lysosomal enzymes.  相似文献   

18.
Phosphomannosyl residues present on lysosomal enzymes are specifically recognized by the mannose 6-phosphate receptor protein. This interaction results in the selective targeting of lysosomal enzymes to lysosomes. While this pathway is operative in many cell types, we have found four cultured cell lines that are deficient in the ability to bind lysosomal enzymes containing phosphomannosyl residues to their intracellular or surface membranes (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). These cells appear to segregate lysosomal enzymes by an alternate intracellular pathway. To determine the basis for the lack of mannose 6-phosphate receptor activity in these cell lines, we studied the biosynthesis of the receptor in receptor-positive (BW5147) and receptor-deficient (P388D1 and MOPC 315) cells. The cells were labeled with [2-3H]mannose or [35S]methionine and the receptor was immunoprecipitated with an antireceptor antiserum. BW5147 cells synthesize a receptor protein whose size increases after translation/glycosylation. MOPC 315 cells produce an apparently normal receptor and degrade it rapidly. P388D1 cells fail to synthesize any detectable receptor. The receptor from BW5147 and MOPC 315 cells is a glycoprotein with both high mannose and complex asparagine-linked oligosaccharides. The complex-type units become fully sialylated and remain so during long periods of chase.  相似文献   

19.
The signal for rapid internalization of the mannose 6-phosphate/insulin-like growth factor II receptor has been localized to the sequence Tyr-Lys-Tyr-Ser-Lys-Val in positions 24-29 of its 163-residue cytoplasmic tail. Most of the activity of this signal is mediated by the carboxyl 4 amino acids, especially Tyr26 and Val29 (Canfield, W. M., Johnson, K. F., Ye, R. D., Gregory, W. and Kornfeld, S. (1991) J. Biol. Chem. 266, 5682-5688). In this study, we have tested the effect of a series of mutations on the internalization rate of a mutant receptor that contains a 29-amino acid cytoplasmic tail terminating with the 4-amino acid internalization sequence Tyr-Ser-Lys-Val. Replacement of Tyr26 with Phe or Trp gave rise to mutant receptors that were internalized at 10% the wild-type rate, while receptors with Ala, Leu, Ile, Val, or Asn at this position were totally inactive. Val29 could be replaced by other large hydrophobic residues (Phe, Leu, Ile, or Met) with no loss of activity, but the presence of Ala, Gly, Arg, Gln, or Tyr in this position inactivated the signal. Ser27 could be effectively replaced by many different amino acids, but not by Pro or Gly. However, Gly27 could be tolerated if the residues at positions 28 and 29 were also changed. A change in the 2-residue spacing between Tyr26 and Val29 destroyed the signal. These data show that the essential elements of this signal are an aromatic residue, especially a Tyr in the first position, separated from a large hydrophobic residue in the last position by 2 amino acids. The residues in positions 2 and 3 of the signal may have a modulating effect on its activity. The Tyr-Ser-Lys-Val signal could be moved to a more proximal region of the cytoplasmic tail with only a modest loss of activity. In addition, the signal could be effectively replaced by the putative 4-residue signals of seven other receptors and membrane proteins known to undergo rapid endocytosis, including the Tyr-Thr-Arg-Phe sequence of the transferrin receptor, a Type II membrane protein. These results are compatible with the 4-residue signals of this type being interchangeable, even among Type I and Type II membrane proteins.  相似文献   

20.
A fusion protein, consisting of the N-terminal 81 amino acids from an inactive bovine DNase I (Q38,E39-E38,Q39) and two sequential synthetic IgG-binding domains based upon domain B of Protein A from Staphylococcus aureus has been shown to bind to porcine IgG with a similar affinity and pH profile to Protein A. The same residue in each B domain (Tyr111 and Tyr169) has been mutated by cassette mutagenesis to Ser, Glu, His, Lys or Arg and the effect of the mutation on binding interactions with porcine IgG investigated. The evidence presented suggests that the interactions at the B domain are highly sensitive to the presence of a charged residue.  相似文献   

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