Isolation and sequencing of a cDNA clone encoding rat liver lysosomal cathepsin D and the structure of three forms of mature enzymes

We isolated and sequenced a cDNA clone corresponding to the entire coding sequence of rat liver lysosomal cathepsin D. The deduced amino acid sequence revealed that cathepsin D consists of 407 amino acid residues (Mr 44,608) and the 20 NH2-terminal residues seem to constitute a cleavable signal peptide after which 44 amino acid residues follow as a propeptide. Two putative N-linked glycosylation sites and aspartic acid in the active site are as well conserved as those of human lysosomal cathepsin D. In the NH2-terminal sequence analysis of two isolated heavy chains of the mature enzyme, the termini were assigned as tryptophan (118th residue) and glycine (165th or 166th residue), respectively, hence demonstrates that the two heavy chains derive from a split of the single chain of cathepsin D at position between 117th and 118th or between 164th and 165th or 165th and 166th amino acids. We conclude that cathepsin D in rat liver lysosomes is a mixture of three forms composed of a single and two two-chain forms. However, the amounts of the two two-chain forms are low compared with that of the single chain form. Densidometric determination after SDS-PAGE revealed that the two two-chain forms account for less than 5% of the single chain form. There is a 82% similarity in amino acid level between rat and human liver lysosomal cathepsin D.     

Human lysosomal acid phosphatase: cloning, expression and chromosomal assignment.

A 2112-bp cDNA clone (lambda CT29) encoding the entire sequence of the human lysosomal acid phosphatase (EC 3.1.3.2) was isolated from a lambda gt11 human placenta cDNA library. The cDNA hybridized with a 2.3-kb mRNA from human liver and HL-60 promyelocytes. The gene for lysosomal acid phosphatase was localized to human chromosome 11. The cDNA includes a 12-bp 5' non-coding region, an open reading frame of 1269 bp and an 831-bp 3' non-coding region with a putative polyadenylation signal 25 bp upstream of a 3' poly(A) tract. The deduced amino acid sequence reveals a putative signal sequence of 30 amino acids followed by a sequence of 393 amino acids that contains eight potential glycosylation sites and a hydrophobic region, which could function as a transmembrane domain. A 60% homology between the known 23 N-terminal amino acid residues of human prostatic acid phosphatase and the N-terminal sequence of lysosomal acid phosphatase suggests an evolutionary link between these two phosphatases. Insertion of the cDNA into the expression vector pSVL yielded a construct that encoded enzymatically active acid phosphatase in transfected monkey COS cells.     

Sequence analysis, tissue distribution, and expression of rat cathepsin S.

Cysteine proteases are involved in many diverse cellular processes ranging from processing of precursor proteins to intracellular degradation. In an effort to identify novel cysteine proteases, we used the polymerase chain reaction and primers directed against the catalytic sites of previously cloned cysteine proteases. From rat brain mRNA, a 600-base pair band was amplified; cloning and partial sequence analysis of this band resulted in the identification of cathepsins B and L and five novel sequences. The novel cDNAs contained a number of residues conserved in lysosomal cysteine proteases, including the active site residue His159 (papain numbering). In addition, the amino acid homology between the novel sequences and either cathepsins B, L, or H, ranged from 63 to 32%. The insert with highest homology was used to screen a rat brain cDNA library; a 1334-base pair cDNA was isolated and the nucleotide sequence determined. This sequence encodes an open reading frame of 330 amino acids which is 82% homologous to human cathepsin S, suggesting that this sequence represents rat cathepsin S. Northern blot analysis for rat cathepsin S revealed tissue-specific expression distinct from the distribution of cathepsin B and L. The regulation of expression of rat cathepsin S mRNA in response to thyroid-stimulating hormone was studied in a rat thyroid cell line FRTL-5. The level of cathepsin S mRNA was substantially increased in response to thyroid-stimulating hormone, whereas cathepsin B and cathepsin L mRNA levels were not altered by this treatment. A portion of cDNA encoding the predicted mature protein of rat cathepsin S was expressed as a glutathione S-transferase-fusion protein. The affinity-purified protein exhibited proteolytic activity with properties similar to bovine cathepsin S. Taken together, these results imply highly specific functions for cathepsin S.     

Purification and characterization of a lysosomal aspartic protease with cathepsin D activity from the mosquito

A lysosomal aspartic protease with cathepsin D activity, from the mosquito, Aedes aegypti, was purified and characterized. Its isolation involved ammonium sulfate (30–50%) and acid (pH 2.5) precipitations of protein extracts from whole previtellogenic mosquitoes followed by cation exchange chromatography. Purity of the enzyme was monitored by SDS-PAGE and silver staining of the gels. The native molecular weight of the purified enzyme as determined by polyacrylamide gel electrophoresis under nondenaturing conditions was 80,000. SDS-PAGE resolved the enzyme into a single polypeptide with M_r = 40,000 suggesting that it exists as a homodimer in its non-denatured state. The pI of the purified enzyme was 5.4 as determined by isoelectric focusing gel electrophoresis. The purified enzyme exhibits properties characteristic of cathepsin D. It utilizes hemoglobin as a substrate and its activity is completely inhibited by pepstatin-A and 6M urea but not by 10 mM KCN. Optimal activity of the purified mosquito aspartic protease was obtained at pH 3.0 and 45°C. With hemoglobin as a substrate the enzyme had an apparent K_m of 4.2 μ M. Polyclonal antibodies to the purified enzyme were raised in rabbits. The specificity of the antibodies to the enzyme was verified by immunoblot analysis of crude mosquito extracts and the enzyme separated by both non-denaturing and SDS-PAGE. Density gradient centrifugation of organelles followed by enzymatic and immunoblot analyses demonstrated the lysosomal nature of the purified enzyme. The N-terminal amino acid sequence of the purified mosquito lysosomal protease (19 amino acids) has 74% identity with N-terminal amino acid sequence of porcine and human cathepsins D.     

Phosphorylation of Asn-linked oligosaccharides located at novel sites on the lysosomal enzyme cathepsin D.

We have examined the phosphorylation of Asn-linked oligosaccharides introduced at seven novel sites on human cathepsin D to determine whether the location of an oligosaccharide on a lysosomal enzyme affects its ability to serve as a substrate for UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (phosphotransferase), the enzyme that catalyzes the initial step in the biosynthesis of mannose 6-phosphate residues. The glycosylation sites were introduced into the cathepsin D cDNA by site-directed mutagenesis and were selected to be widely distributed over the surface of the molecule. When the constructs were expressed in Xenopus oocytes, the oligosaccharides at each glycosylation site were phosphorylated at levels considerably above background (19-70% phosphorylation versus < 0.4% for the secretory protein glycopepsinogen). However, oligosaccharides located closer to the essential components of the phosphotransferase recognition domain (lysine 203 and amino acids 265-292) were phosphorylated better than oligosaccharides located further away. Similar results were obtained for oligosaccharides at homologous sites on a pepsinogen/cathepsin D chimera containing only lysine 203 and residues 265-319 of cathepsin D, although the absolute levels of phosphorylation were lower. These results demonstrate that there is considerable flexibility in the placement of glycosylation sites on cathepsin D in terms of the ability of the oligosaccharides to serve as substrates for phosphotransferase, although oligosaccharides located closer to the phosphotransferase recognition determinant are preferentially phosphorylated.     

Isolation and sequencing of a cDNA clone encoding the 85 kDa human lysosomal sialoglycoprotein (hLGP85) in human metastatic pancreas islet tumor cells.

A full length cDNA for a human lysosomal membrane sialoglycoprotein (hLGP85) was isolated as a probe of the cDNA of rat LGP85 (rLGP85) from the cDNA library prepared from total mRNA of QGP-1NL cells, a human pancreatic islet tumor cell with a high metastatic activity. The deduced amino acid sequence shows that hLGP85 consists of 478 amino acid residues (MW. 54,289). The protein has 10 putative N-glycosylation sites and 2 hydrophobic regions at the NH2- and near the COOH-termini, respectively. Thus, both domains probably constitute putative transmembrane domains. It exhibits 86% and 79% sequence similarities in amino acids and nucleic acids to rat lysosomal membrane sialoglycoprotein (rLGP85), respectively. The protein contained the short cytoplasmic tail at the COOH-terminus which does not form the glycine-tyrosine sequence (GY motif), the so-called lysosomal targetting signal.     

Molecular cloning of rat precursor cathepsin H and the expression of five lysosomal cathepsins in normal tissues and in a rat carcinosarcoma

1. A rat cathepsin H cDNA was isolated from a rat liver cDNA library with synthetic oligonucleotide probes. 2. DNA sequence analysis indicated that it codes for rat preprocathepsin H. 3. Using this clone together with the cDNA for cathepsins B, D, L and S as probes, the expression of five major lysosomal proteinases was investigated in ten different normal rat tissues and in a rat carcinoma. 4. The common feature of their expression is that the five cathepsins have relatively high mRNA levels in lung and kidney, suggesting that they all play important roles in organs engaged in active protein metabolism. 5. In other tissues, the concentrations of the five cathepsin mRNAs are significantly different. This may indicate that their expressions are differentially regulated and that they may have specialized functions in specific tissues. 6. The cathepsin B mRNA level is at least 2.5-fold higher in the rat W256-carcinoma than in any of the normal rat tissues surveyed. 7. In contrast, the mRNA levels for the other four cathepsins show no comparable elevations. 8. This finding is consistent with previous observations reporting a correlation between cathepsins B expression and malignant tumors.     

Primary structure and processing of lysosomal alpha-glucosidase; homology with the intestinal sucrase-isomaltase complex.

Lysosomal alpha-glucosidase (acid maltase) is essential for degradation of glycogen in lysosomes. Enzyme deficiency results in glycogenosis type II. The amino acid sequence of the entire enzyme was derived from the nucleotide sequence of cloned cDNA. The cDNA comprises 3636 nt, and hybridizes with a messenger RNA of approximately 3.6 kb, which is absent in fibroblasts of two patients with glycogenosis type II. The encoded protein has a molecular mass of 104.645 kd and starts with a signal peptide. Sites of proteolytic processing are established by identification of N-terminal amino acid sequences of the 110-kd precursor, and the 76-kd and 70-kd mature forms of the enzyme encoded by the cDNA. Interestingly, both amino-terminal and carboxy-terminal processing occurs. Sites of sugar-chain attachment are proposed. A remarkable homology is observed between this soluble lysosomal alpha-glucosidase and the membrane-bound intestinal brush border sucrase-isomaltase enzyme complex. It is proposed that these enzymes are derived from the same ancestral gene. Around the putative active site of sucrase and isomaltase, 10 out of 13 amino acids are identical to the corresponding amino acids of lysosomal alpha-glucosidase. This strongly suggests that the aspartic acid residue at this position is essential for catalytic function of lysosomal alpha-glucosidase.     

A His-Leu-Leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor is necessary for the lysosomal enzyme sorting function.

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.     

The primary structure of mouse saposin.

The primary structure of mouse sphingolipid activator protein (saposin) was determined by cDNA sequencing. The amino acid sequence predicted by the cDNA sequence revealed that mouse saposin was highly homologous to human saposin and also to rat sertoli cell glycoprotein. Mouse saposin also has four functional domains, which are structurally similar to each other, and each domain has cysteines, prolines, and a potential glycosylation site at an almost identical position. An amino acid comparison between human and mouse saposins revealed that the similarity was approximately 70%, and human saposin lacks thirty-one amino acids between domains C and D. Heterogeneities of mRNA were found in both the coding and noncoding regions.     

The cDNA sequence of mouse LAMP-2. Evidence for two classes of lysosomal membrane glycoproteins

We describe the isolation and sequencing of a cDNA encoding the mouse lysosomal membrane glycoprotein mLAMP-2 and the sequence differences that distinguish this molecule from the LAMP-1 class of proteins. An oligonucleotide probe corresponding to the NH2-terminal amino acid sequence of purified mLAMP-2 was synthesized by the polymerase chain reaction and used to screen several cDNA libraries. cDNA clones with an insert of 1,700 nucleotides were identified and sequenced. The deduced amino acid sequence of mLAMP-2 comprises a signal sequence of 25 residues and a 390-amino acid polypeptide (Mr 43,017) with the following putative domains: a large intraluminal region (residues 1-354) with 17 N-linked glycosylation sites (Asn-X-Ser/Thr), a hydrophobic transmembrane-spanning region of 24 residues (355-378), and a COOH-terminal cytoplasmic tail of 12 residues (379-390). When this sequence is compared with those of other lysosomal membrane glycoproteins, it is apparent that mouse LAMP-2 and human LAMP-2 form one homology class (LAMP-2) that is separated from the LAMP-1 class of proteins. The sequence differences in these two classes provide a basis for comparing the structure of the proteins with their biochemical and biological properties.     

Isolation and sequencing of a cDNA clone encoding 96 kDa sialoglycoprotein in rat liver lysosomal membranes

We isolated and sequenced LGP 96, a cDNA clone corresponding to the entire coding sequence of the rat liver lysosomal membrane sialoglycoprotein with an apparent Mr of 96 K, LGP 96. The deduced amino acid sequence indicates that LGP 96 consists of 411 amino acid residues (Mr 45,163) and the 26 NH2-terminal residues presumably constitute a cleavable signal peptide. The major portion of LGP 96 resides on the luminal side of the lysosome and bears a large number of N-linked heavily sialylated complex type carbohydrate chains, giving the mature molecule of 96 kDa. The protein has 17 potential N-glycosylation sites and 32.1 and 65.3% sequence similarities in amino acid to LGP 107 and human lamp-2, respectively. The glycosylation sites are clustered into two domains separated by a hinge-like structure enriched with proline and threonine. LGP 96 possesses one putative transmembrane domain consisting of 24 hydrophobic amino acids near the COOH-terminus and contains a short cytoplasmic segment constituting 12 amino acid residues at the COOH-terminal end. Comparison of LGP 96 and recently cloned lysosomal membrane glycoprotein sequences reveals strong similarity in the putative transmembrane domain and cytoplasmic tail. It is very likely that these portions are important for the targeting of molecules to lysosomes. A comparison of LGP 96 and LGP 107 showed numerous structural similarities.     

Identification of a novel transforming growth factor-beta (TGF-beta 5) mRNA in Xenopus laevis

A novel transforming growth factor-beta (TGF-beta) mRNA of about 3.0 kilobases, which encodes a putative protein of 382 amino acids, has been identified in amphibians by cDNA cloning. This mRNA, which we designate as TGF-beta 5, is developmentally regulated and highly expressed beginning at early neurula (stage 14) and in many adult tissues in Xenopus laevis. Following the first methionine, the putative precursor protein has a hydrophobic region, approximately 22 amino acids long, which probably represents a signal sequence, similar to that found in TGF-beta s 1-3. The precursor also has potential sites for glycosylation, integrin binding (RGD), and a tetrabasic amino acid (RKKR) site for potential cleavage of the precursor peptide to a biologically active protein. The putative mature protein consists of 112 amino acids with 9 cysteines and has 76, 66, 69, and 72% identity to TGF-beta s 1-4, respectively.     

Mouse cathepsin M, a placenta-specific lysosomal cysteine protease related to cathepsins L and P

The complete nucleotide sequence of a novel cathepsin cDNA derived from mouse placenta was determined and is termed cathepsin M. The predicted protein of 333 amino acid is a member of the family C1A proteases and is related to mouse cathepsins L and P. Mouse cathepsin M is highly expressed in placenta, whereas no detectable levels were found in lung, spleen, heart, brain, kidney, thymus, testicle, liver, or embryo. Phylogenic analyses of the sequences of human and mouse cathepsins show that cathepsin M is most closely related to cathepsins P and L. However, the differences are sufficiently large to indicate that the enzymes will be found in other species. This is in contrast to human cathepsins L and V, which probably resulted from a gene duplication after divergence of mammalian species.     

Biosynthesis of lysosomal endopeptidases

Despite the clear differences between the amino acid sequence and enzymatic specificity of aspartic and cysteine endopeptidases, the biosynthetic processing of lysosomal members of these two families is very similar. With in vitro translation and pulse-chase analysis in tissue culture cells, the biosynthesis of cathepsin D, a aspartic protease, and cathepsins B, H and L, cysteine proteases, are compared. Both aspartic and cysteine endopeptidases undergo cotranslational cleavage of an amino-terminal signal peptide that mediates transport across the endoplasmic reticulum (ER) membrane. Addition of high-mannose carbohydrate also occurs cotranslationally in the lumen of the ER. Proteases of both enzyme classes are initially synthesized as inactive proenzymes possessing amino-terminal activation peptides. Removal of the propeptide generates an active single-chain enzyme. Whether the single-chain enzyme undergoes asymmetric cleavage into a light and a heavy chain appears to be cell type specific. Finally, late during their biosynthesis both classes of enzymes undergo amino acid trimming, losing a few amino acid residues at the cleavage site between the light and heavy chains and/or at their carboxyltermini. During biosynthesis these enzymes are also secreted to some extent. In most cells the secreted enzyme is the proenzyme bearing some complex carbohydrate. Under certain physiological conditions the inactive secreted enzymes may become activated as a result of a conformational change that may or may not result in autolysis. Analysis of the biochemical nature of the various processing steps helps define the cellular pathway followed by newly synthesized proteases targeted to the lysosome.     

Purification, cDNA cloning, and expression of a new human blood plasma glutamate carboxypeptidase homologous to N-acetyl-aspartyl-alpha-glutamate carboxypeptidase/prostate-specific membrane antigen

We describe the identification, cDNA cloning, and biochemical characterization of a new human blood plasma glutamate carboxypeptidase (PGCP). PGCP was co-purified from human placenta with lysosomal carboxypeptidase, cathepsin A, lysosomal endopeptidase, cathepsin D, and a gamma-interferon-inducible protein, IP-30, using an affinity chromatography on a Phe-Leu-agarose column. A PGCP cDNA was obtained as an expressed sequence tag clone and completed at 5'-end by rapid amplification of cDNA ends polymerase chain reaction. The cDNA contained a 1623-base pair open reading frame predicting a 541-amino acid protein, with five putative Asn glycosylation sites and a 21-residue signal peptide. PGCP showed significant amino acid sequence homology to several cocatalytic metallopeptidases including a glutamate carboxypeptidase II also known as N-acetyl-aspartyl-alpha-glutamate carboxypeptidase or as prostate-specific membrane antigen and expressed glutamate carboxypeptidase activity. Expression of the PGCP cDNA in COS-1 cells, followed by Western blotting and metabolic labeling showed that PGCP is synthesized as a 62-kDa precursor, which is processed to a 56-kDa mature form containing two Asn-linked oligosaccharide chains. The mature form of PGCP was secreted into the culture medium, which is consistent with its intracellular localization in secretion granules. In humans, PGCP is found principally in blood plasma, suggesting a potential role in the metabolism of secreted peptides.     

Isolation and sequencing of a cDNA clone encoding acid phosphatase in rat liver lysosomes

A full length cDNA for acid phosphatase in rat liver lysosomes was isolated and sequenced. The predicted amino acid sequence comprises 423 residues (48,332 Da). A putative signal peptide of 30 residues is followed by the NH2-terminal sequence of lysosomal acid phosphatase (45,096 Da). The deduced NH2-terminal 18-residue sequence is identical with that determined directly for acid phosphatases purified from the rat liver lysosomal membranes. The primary structure deduced for acid phosphatase contains 9 potential N-glycosylation sites and a hydrophobic region which could function as a transmembrane domain. It exhibits 89% and 67% sequence similarities in amino acids and nucleic acids, respectively, to human lysosomal acid phosphatase. The amino acid sequence of the putative transmembrane segment shows a complete similarity to that of the human enzyme. Northern blot hybridization analysis identified a single species of acid phosphatase mRNA (2.2 kbp in length) in rat liver.