Gene_locus Report for: human-PRCP

Prolylcarboxypeptidase, a lysosomal serine carboxypeptidase, cleaves COOH-terminal amino acids linked to proline, as in angiotensin II and III and [des-Arg9] bradykinin. About 25% of the enzyme protein was sequenced, and the complete sequence was deduced from its human kidney cDNA. The cDNA insert contained an open reading frame of 1488 base pairs coding for a protein of 496 residues. The authentic NH2-terminal sequence matched the deduced protein sequence starting with residue 46, suggesting the presence of both a signal and propeptide. The mature enzyme (451 residues) has a calculated M(r) = 51,043, whereas the M(r) of the purified glycoprotein is 58,000, indicating 12% carbohydrate. The overall sequence identity with serine peptidases is low (10-18%), but sequences around residues of the putative catalytic triad (Ser134, Asp333, His411) are similar (30-67%) to both the serine carboxypeptidases (e.g. deamidase or lysosomal protective protein, yeast carboxypeptidase Y, and KEX1 gene product) and the prolylendopeptidase family. Thus, prolylcarboxypeptidase links these two families, suggesting an evolutionary relationship. It is inhibited (Ki = 2.6 x 10(-7) M) by benzyloxycarbonyl-Pro-prolinal, a specific inhibitor of prolylendopeptidase, another angiotensin metabolizing enzyme. Prolylcarboxypeptidase contains serine or threonine residues repeated as the 26th residue 7 out of 9 times, with identical or similar amino acids in other positions in the repeats. The KEX1 gene product contains a similar motif, with serine or threonine as every 27th residue. The importance of prolylcarboxypeptidase is strongly suggested by its presence in various organs and cells and by the substrates it cleaves.

Prolylcarboxypeptidase was purified from human kidney 1200-fold with 18% yield. The enzyme had no cathepsin A activity and appeared to be homogeneous in gel electrophoresis. The molecular weight of prolylcarboxypeptidase was estimated to be 115,000 by gel filtration. Under denaturing conditions the enzyme dissociated into subunits of 45,000 and 66,500 molecular weight. The enzyme cleaved benzyloxycarbonyl (Cbz)-Pro-Phe, representing the COOH-terminal end of angiotensin II and des-Asp1-angiotensin II (angiotensin III), at a rate of 31 micronmol/h/mg of protein. The rate of hydrolysis increased when phenylalanine in the N-protected dipeptide was replaced with alanine, valine, or leucine or when the octapeptide angiotensin II or the heptapeptide angiotensin III were the substrates. The enzyme also cleaved the angiotensin II antagonist saralasin (Sar1-Ala8-angiotensin II). The Km values were 1 mM, 2mM, and 0.77 mM with Cbz-Pro-Phe, angiotensin II, and angiotensin III, respectively. The enzyme had an acid pH optimum (4.5 to 5.5), but hydrolyzed angiotensin III at pH 7 at 50% of the optimal rate. Prolylcarboxypeptidase was inhibited by diisopropyl phosphorofluoridate and pepstatin, but not by sequestering agents or -SH reagents.

Prolylcarboxypeptidase (PrCP) is a lysosomal serine carboxypeptidase that cleaves a variety of C-terminal amino acids adjacent to proline and has been implicated in diseases such as hypertension and obesity. Here, the robust production, purification and crystallization of glycosylated human PrCP from stably transformed CHO cells is described. Purified PrCP yielded crystals belonging to space group R32, with unit-cell parameters a = b = 181.14, c = 240.13 A, that diffracted to better than 2.8 A resolution.

BACKGROUND: The unique S28 family of proteases is comprised of the carboxypeptidase PRCP and the aminopeptidase DPP7. The structural basis of the different substrate specificities of the two enzymes is not understood nor has the structure of the S28 fold been described. RESULTS: The experimentally phased 2.8 A crystal structure is presented for human PRCP. PRCP contains an alpha/beta hydrolase domain harboring the catalytic Asp-His-Ser triad and a novel helical structural domain that caps the active site. Structural comparisons with prolylendopeptidase and DPP4 identify the S1 proline binding site of PRCP. A structure-based alignment with the previously undescribed structure of DPP7 illuminates the mechanism of orthogonal substrate specificity of PRCP and DPP7. PRCP has an extended active-site cleft that can accommodate proline substrates with multiple N-terminal residues. In contrast, the substrate binding groove of DPP7 is occluded by a short amino-acid insertion unique to DPP7 that creates a truncated active site selective for dipeptidyl proteolysis of N-terminal substrates. CONCLUSION: The results define the structure of the S28 family of proteases, provide the structural basis of PRCP and DPP7 substrate specificity and enable the rational design of selective PRCP modulators.

Chromosome 11, although average in size, is one of the most gene- and disease-rich chromosomes in the human genome. Initial gene annotation indicates an average gene density of 11.6 genes per megabase, including 1,524 protein-coding genes, some of which were identified using novel methods, and 765 pseudogenes. One-quarter of the protein-coding genes shows overlap with other genes. Of the 856 olfactory receptor genes in the human genome, more than 40% are located in 28 single- and multi-gene clusters along this chromosome. Out of the 171 disorders currently attributed to the chromosome, 86 remain for which the underlying molecular basis is not yet known, including several mendelian traits, cancer and susceptibility loci. The high-quality data presented here--nearly 134.5 million base pairs representing 99.8% coverage of the euchromatic sequence--provide scientists with a solid foundation for understanding the genetic basis of these disorders and other biological phenomena.

As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

Prolylcarboxypeptidase, a lysosomal serine carboxypeptidase, cleaves COOH-terminal amino acids linked to proline, as in angiotensin II and III and [des-Arg9] bradykinin. About 25% of the enzyme protein was sequenced, and the complete sequence was deduced from its human kidney cDNA. The cDNA insert contained an open reading frame of 1488 base pairs coding for a protein of 496 residues. The authentic NH2-terminal sequence matched the deduced protein sequence starting with residue 46, suggesting the presence of both a signal and propeptide. The mature enzyme (451 residues) has a calculated M(r) = 51,043, whereas the M(r) of the purified glycoprotein is 58,000, indicating 12% carbohydrate. The overall sequence identity with serine peptidases is low (10-18%), but sequences around residues of the putative catalytic triad (Ser134, Asp333, His411) are similar (30-67%) to both the serine carboxypeptidases (e.g. deamidase or lysosomal protective protein, yeast carboxypeptidase Y, and KEX1 gene product) and the prolylendopeptidase family. Thus, prolylcarboxypeptidase links these two families, suggesting an evolutionary relationship. It is inhibited (Ki = 2.6 x 10(-7) M) by benzyloxycarbonyl-Pro-prolinal, a specific inhibitor of prolylendopeptidase, another angiotensin metabolizing enzyme. Prolylcarboxypeptidase contains serine or threonine residues repeated as the 26th residue 7 out of 9 times, with identical or similar amino acids in other positions in the repeats. The KEX1 gene product contains a similar motif, with serine or threonine as every 27th residue. The importance of prolylcarboxypeptidase is strongly suggested by its presence in various organs and cells and by the substrates it cleaves.

Prolylcarboxypeptidase was purified from human kidney 1200-fold with 18% yield. The enzyme had no cathepsin A activity and appeared to be homogeneous in gel electrophoresis. The molecular weight of prolylcarboxypeptidase was estimated to be 115,000 by gel filtration. Under denaturing conditions the enzyme dissociated into subunits of 45,000 and 66,500 molecular weight. The enzyme cleaved benzyloxycarbonyl (Cbz)-Pro-Phe, representing the COOH-terminal end of angiotensin II and des-Asp1-angiotensin II (angiotensin III), at a rate of 31 micronmol/h/mg of protein. The rate of hydrolysis increased when phenylalanine in the N-protected dipeptide was replaced with alanine, valine, or leucine or when the octapeptide angiotensin II or the heptapeptide angiotensin III were the substrates. The enzyme also cleaved the angiotensin II antagonist saralasin (Sar1-Ala8-angiotensin II). The Km values were 1 mM, 2mM, and 0.77 mM with Cbz-Pro-Phe, angiotensin II, and angiotensin III, respectively. The enzyme had an acid pH optimum (4.5 to 5.5), but hydrolyzed angiotensin III at pH 7 at 50% of the optimal rate. Prolylcarboxypeptidase was inhibited by diisopropyl phosphorofluoridate and pepstatin, but not by sequestering agents or -SH reagents.