Re-entering the translocon from the lumenal side of the endoplasmic reticulum. Studies on mutated carboxypeptidase yscY species. (1/1719)

Misfolded or unassembled secretory proteins are retained in the endoplasmic reticulum (ER) and subsequently degraded by the cytosolic ubiquitin-proteasome system. This requires their retrograde transport from the ER lumen into the cytosol, which is mediated by the Sec61 translocon. It had remained a mystery whether ER-localised soluble proteins are at all capable of re-entering the Sec61 channel de novo or whether a permanent contact of the imported protein with the translocon is a prerequisite for retrograde transport. In this study we analysed two new variants of the mutated yeast carboxypeptidase yscY, CPY*: a carboxy-terminal fusion protein of CPY* and pig liver esterase and a CPY* species carrying an additional glycosylation site at its carboxy-terminus. With these constructs it can be demonstrated that the newly synthesised CPY* chain is not retained in the translocation channel but reaches its ER lumenal side completely. Our data indicate that the Sec61 channel provides the essential pore for protein transport through the ER membrane in either direction; persistent contact with the translocon after import seems not to be required for retrograde transport.  (+info)

A soluble form of the avian hepatitis B virus receptor. Biochemical characterization and functional analysis of the receptor ligand complex. (2/1719)

Avian hepatitis B virus infection is initiated by the specific interaction of the extracellular preS part of the large viral envelope protein with carboxypeptidase D (gp180), the primary cellular receptor. To functionally and biochemically characterize this interaction, we purified a soluble form of duck carboxypeptidase D from a baculovirus expression system, confirmed its receptor function, and investigated the contribution of different preS sequence elements to receptor binding by surface plasmon resonance analysis. We found that preS binds duck carboxypeptidase D with a 1:1 stoichiometry, thereby inducing conformational changes but not oligomerization. The association constant of the complex was determined to be 2.2 x 10(7) M-1 at 37 degreesC, pH 7.4, with an association rate of 4.0 x 10(4) M-1 s-1 and a dissociation rate of 1.9 x 10(-3) s-1, substantiating high affinity interaction of avihepadnaviruses with their receptor carboxypeptidase D. The separately expressed receptor-binding domain, comprising about 50% of preS as defined by mutational analysis, exhibits similar constants. The domain consists of an essential element, probably responsible for the initial receptor contact and a part that contributes to complex stabilization in a conformation sensitive manner. Together with previous results from cell biological studies these data provide new insights into the initial step of hepadnaviral infection.  (+info)

Properties of non-polymerizable tropomyosin obtained by carboxypeptidase A digestion. (3/1719)

Tropomyosin digested with carboxypeptidase A [EC] (CTM) shows a lower viscosity than the undigested protein in solution. From the relation between the viscosity decrease and the amount of amino acids liberated from the carboxyl terminus during this digestion, it is inferred that loss of the tri-peptide-Thr-Ser-Ile from the C-terminus is responsible for the decrease in viscosity. The secondary structure of -TM was not affected by the digestion according to circular dichroic measurements. The viscosity of CTM did not increase in methanol-water mixtures, whereas that of tropomyosin increased markedly. These results indicate that polymerizability was lost upon the removal of a small peptide from the C-terminus without change in the secondary structure. A decrease in the viscosity of tropomyosin solutions was observed on the addition of CTM, indicating that CTM interacts with intact tropomyosin. The dependence of the viscosity decrease on the amount of CTM showed that CTM binds tropomyosin in a one-to-one ratio as a result of end-to-end interaction. Since paracrystals having a 400 A repeated band structure could be grown in the presence of Mg ions at neutral pH, side-by-side interactions in CTM molecules remain intact, even though polymerizability is lost. The disc gel electrophoretic pattern showed that troponin could bind to CTM, but no increase in viscosity due to the complex was observed in solution. That is, the C-terminal part of tropomyosin is not required for the formation of the complex. The amount of CTM bound to F-actin was less than half of that bound to undigested tropomyosin, and could be reduced to one-tenth by a washing procedure. In the presence of troponin, however, the amount recovered to the level of tropomyosin normally bound to F-actin. Therefore, it is concluded that troponin is bound in the middle of the tropomyosin molecule and strengthens the binding of tropomyosin to F-actin.  (+info)

Mutational analysis of active-site residues of the enterococcal D-ala-D-Ala dipeptidase VanX and comparison with Escherichia coli D-ala-D-Ala ligase and D-ala-D-Ala carboxypeptidase VanY. (4/1719)

BACKGROUND: Vancomycin-resistant enterococci are pathogenic bacteria that attenuate antibiotic sensitivity by producing peptidoglycan precursors that terminate in D-Ala-D-lactate rather than D-Ala-D-Ala. A key enzyme in effecting antibiotic resistance is the metallodipeptidase VanX, which reduces the cellular pool of the D-Ala-D-Ala dipeptide. RESULTS: We constructed eleven mutants, using the recently determined VanX structure as a basis, to investigate residue function. Mutating Asp142 or Ser114 showed a large effect principally on KM, consistent with roles in recognition of the D-Ala-D-Ala termini. The drastic reduction or absence of activity in the Arg71 mutants correlates with a role in the stabilization of an anionic tetrahedral transition state. Three residues of the Escherichia coli D-Ala-D-Ala ligase (Ddl), Glu15, Ser 281 and Arg255, are similarly conserved and have equivalent functions with respect to VanX, consistent with a convergent evolution of active sites to bind D-Ala-D-Ala and lower energy barriers for formation of the tetrahedral intermediate and transition states. In the N-acyl-D-Ala-D-Ala carboxypeptidase VanY, all active-site residues are conserved (except for the two responsible for recognition of the dipeptide amino terminus). CONCLUSIONS: The mutagenesis results support structure-based functional predictions and explain why the VanX dipeptidase and Ddl ligase show narrow specificity for the D,D-dipeptide substrate. The results reveal that VanX and Ddl, two enzymes that use the same substrate but proceed in opposite directions driven by distinct cofactors (zinc versus ATP), evolved similar architectural solutions to substrate recognition and catalysis acceleration. VanY sequence analysis predicts an active site and mechanism of reaction similar to VanX.  (+info)

Isolation and expression of novel human glutamate carboxypeptidases with N-acetylated alpha-linked acidic dipeptidase and dipeptidyl peptidase IV activity. (5/1719)

Hydrolysis of the neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) by N-acetylated alpha-linked acidic dipeptidase (NAALADase) to release glutamate may be important in a number of neurodegenerative disorders in which excitotoxic mechanisms are implicated. The gene coding for human prostate-specific membrane antigen, a marker of prostatic carcinomas, and its rat homologue glutamate carboxypeptidase II have recently been shown to possess such NAALADase activity. In contrast, a closely related member of this gene family, rat ileal 100-kDa protein, possesses a dipeptidyl peptidase IV activity. Here, we describe the cloning of human ileal 100-kDa protein, which we have called a NAALADase- "like" (NAALADase L) peptidase based on its sequence similarity to other members of this gene family, and its inability to hydrolyze NAAG in transient transfection experiments. Furthermore, we describe the cloning of a third novel member of this gene family, NAALADase II, which codes for a type II integral membrane protein and which we have localized to chromosome 11 by fluorescent in situ hybridization analysis. Transient transfection of NAALADase II cDNA confers both NAALADase and dipeptidyl peptidase IV activity to COS cells. Expression studies using reverse transcription-polymerase chain reaction and Northern blot hybridization show that NAALADase II is highly expressed in ovary and testis as well as within discrete brain areas.  (+info)

The protein disulphide-isomerase family: unravelling a string of folds. (6/1719)

The mammalian protein disulphide-isomerase (PDI) family encompasses several highly divergent proteins that are involved in the processing and maturation of secretory proteins in the endoplasmic reticulum. These proteins are characterized by the presence of one or more domains of roughly 95-110 amino acids related to the cytoplasmic protein thioredoxin. All but the PDI-D subfamily are composed entirely of repeats of such domains, with at least one domain containing and one domain lacking a redox-active -Cys-Xaa-Xaa-Cys- tetrapeptide. In addition to their known roles as redox catalysts and isomerases, the last few years have revealed additional functions of the PDI proteins, including peptide binding, cell adhesion and perhaps chaperone activities. Attention is now turning to the non-redox-active domains of the PDIs, which may play an important role in all of the known activities of these proteins. Thus the presence of both redox-active and -inactive domains within these proteins portends a complexity of functions differentially accommodated by the various family members.  (+info)

Cloning, expression, and substrate specificity of MeCPA, a zinc carboxypeptidase that is secreted into infected tissues by the fungal entomopathogen Metarhizium anisopliae. (7/1719)

To date zinc carboxypeptidases have only been found in animals and actinomycete bacteria. A cDNA clone (MeCPA) for a novel fungal (Metarhizium anisopliae) carboxypeptidase (MeCPA) was obtained by using reverse transcription differential display polymerase chain reaction to identify pathogenicity genes. MeCPA resembles pancreatic carboxypeptidases in being synthesized as a precursor species (418 amino acids) containing a large amino-terminal fragment (99 amino acids). The mature (secreted) form of MeCPA shows closest amino acid identity to human carboxypeptidases A1 (35%) and A2 (37%). MeCPA was expressed in an insect cell line yielding an enzyme with dual A1 + A2 specificity for branched aliphatic and aromatic COOH-terminal amino acids. However, in contrast to the very broad spectrum A + B-type bacterial enzymes, MeCPA lacks B-type activity against charged amino acids. This is predictable as key catalytic residues determining the specificity of MeCPA are conserved with those of mammalian A-type carboxypeptidases. Thus, in evolutionary terms the fungal enzyme is an intermediate between the divergence of A and B forms and the differentiation of the A form into A1 and A2 isoforms. Ultrastructural immunocytochemistry of infected host (Manduca sexta) cuticle demonstrated that MeCPA participates with the concurrently produced endoproteases in procuring nutrients; an equivalent function to digestive pancreatic enzymes.  (+info)

Cloning, sequencing and functional expression of a cDNA encoding porcine pancreatic preprocarboxypeptidase A1. (8/1719)

A full-length cDNA clone coding for porcine pancreatic preprocarboxypeptidase A1 (prePCPA1) was isolated from a cDNA library. The open reading frame (ORF) of the nucleotide sequence was 1260 nt in length and encoded a protein of 419 amino acids (aa). The cDNA included a short signal peptide of 16 aa and a 94 aa-long activation segment. The calculated molecular mass of the mature proenzyme was 45561 Da, in accordance with that of the purified porcine pancreatic PCPA1. The deduced aa sequence of the corresponding enzyme differed from that predicted by the three-dimensional structure by 40 aa, and showed 85% identity and 55% identity to that of procarboxypeptidases A1 and A2, respectively. Moreover the sequence was identical to that of several independent cDNA clones, suggesting that it is the major transcribed gene. No evidence for a second variant was observed in the cDNA library and PCPA2 is apparently absent from the porcine pancreas. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast triose phosphate isomerase promoter. The signal peptide of the PCPA protein efficiently directed its secretion into the culture medium (1.5 mg.L-1) as a protein of the predicted size. The recombinant proenzyme was analyzed by immunological and enzymological methods. Its activation behavior was comparable with that of the native form and led to a 35-kDa active enzyme.  (+info)