Human lysosomal protective protein has cathepsin A-like activity distinct from its protective function

The protective protein was first discovered because of its deficiency in the metabolic storage disorder galactosialidosis. It associates with lysosomal beta-galactosidase and neuraminidase, toward which it exerts a protective function necessary for their stability and activity. Human and mouse protective proteins are homologous to yeast and plant serine carboxypeptidases. Here, we provide evidence that this protein has enzymatic activity similar to that of lysosomal cathepsin A: 1) overexpression of human and mouse protective proteins in COS-1 cells induces a 3-4-fold increase of cathepsin A-like activity; 2) this activity is reduced to approximately 1% in three galactosialidosis patients with different clinical phenotypes; 3) monospecific antibodies raised against human protective protein precipitate virtually all cathepsin A-like activity in normal human fibroblast extracts. Mutagenesis of the serine and histidine active site residues abolishes the enzymatic activity of the respective mutant protective proteins. These mutants, however, behave as the wild-type protein with regard to intracellular routing, processing, and secretion. In contrast, modification of the very conserved Cys60 residue interferes with the correct folding of the precursor polypeptide and, hence, its intracellular transport and processing. The secreted active site mutant precursors, endocytosed by galactosialidosis fibroblasts, restore beta-galactosidase and neuraminidase activities as effectively as wild-type protective protein. These findings indicate that the catalytic activity and protective function of the protective protein are distinct.     

Human lysosomal protective protein. Glycosylation, intracellular transport, and association with beta-galactosidase in the endoplasmic reticulum.

In lysosomes beta-galactosidase and neuraminidase acquire a stable and active conformation through their association with the protective protein. The latter is homologous to serine carboxypeptidases and has cathepsin A-like activity which is distinct from its protective function towards the two glycosidases. To define signals in the human protective protein important for its intracellular transport, and to determine the site of its association with beta-galactosidase, we have generated a set of mutated protective protein cDNAs carrying targeted base substitutions. These mutants were either singly transfected into COS-1 cells or cotransfected together with wild type human beta-galactosidase. We show that all point mutations cause either a complete or partial retention of the protective protein precursor in the endoplasmic reticulum. This abnormal accumulation leads to degradation of the mutant proteins probably in this compartment. Only the oligosaccharide chain on the 32-kDa subunit acquires the mannose 6-phosphate recognition marker, the one on the 20-kDa subunit seems to be merely essential for the stability of the mature protein. In cotransfection experiments, wild type beta-galactosidase and protective protein appear to assemble already as precursors, soon after synthesis, in the endoplasmic reticulum. Mutated protective protein precursors that are retained in the endoplasmic reticulum or pre-Golgi complex interact with and withhold normal beta-galactosidase molecules in the same compartments, thereby preventing their normal routing.     

Pycnodysostosis: role and regulation of cathepsin K in osteoclast function and human disease

Patients with pycnodysostosis, a rare skeletal dysplasia, present with bone abnormalities such as short stature, acroosteolysis of distal phalanges, and skull deformities. The disease is caused by a deficiency of the cysteine protease cathepsin K which is responsible for degradation of collagen type I and other bone proteins. Osteoclasts, bone cells of hematopoietic origin responsible for bone mineral as well as protein matrix degradation, are dysfunctional in patients with pycnodysostosis due to mutations in the cathepsin K gene. Cathepsin K deficient osteoclasts can demineralize bone but cannot degrade the protein matrix. Mutations in the cathepsin K gene disrupting wild type cathepsin K activity have been described in patients with pycnodysostosis. Animal models of cathepsin K deficiency have been created and provide a valuable tool to study osteoclast function and treatment for cathepsin K deficiency. Understanding the regulation and role of cathepsin K in osteoclast function is important for designing future therapies for pycnodysostosis. Cathepsin K inhibitors will be useful in pathological processes involving excess osteoclast activation and bone resorption such as osteoporosis, bone metastasis and multiple myeloma. This review will discuss the bone remodeling cycle, the human disease pycnodysostosis caused by cathepsin K deficiency and cathepsin K activity and regulation.     

Expression of lysosomal protective protein/cathepsin A in a stably transformed human neuroblastoma cell line during bi-directional differentiation into neuronal and Schwannian cells

Human neuroblastoma GOTO cell lines were established that stably express recombinant human lysosomal protective protein/cathepsin A (PPCA) cDNA by transfection. Intracellular cathepsin A (acid serine carboxypeptidase) activity increased four-fold compared with in those of the parent and mock-transfected cell lines. The immunoreactive 54 kDa precursor/zymogen and mature 32/20 kDa two-chain forms were produced in the cells. The amount of the latter form expressed in the GOTO cells was significantly larger than those in the PPCA-overexpressing CHO cell lines previously established. The intracellular proteins showed a typical lysosomal granular distribution and the glycosylated 54 kDa precursor was secreted into the culture medium without the addition of an alkalizing agent. The PPCA-overexpressing cell lines also retained the ability to differentiate bi-directionally as well as the parent cells; into neuronal cells on induction by dibutyryl cAMP in serum-free medium and into Schwannian cells on induction by bromodeoxyuridine. During the course of differentiation into neuronal and Schwannian cells, the intracellular cathepsin A activity further increased two and five times, respectively, which was associated with an increase in the expression of the 32/20 kDa two-chain form. The glycosylated precursor proteins were taken up via the mannose 6-phosphate receptors, and the cathepsin A, alpha-neuraminidase and beta-galactosidase (beta-Gal) activities deficient in the fibroblasts derived from a patient with PPCA deficiency (galactosialidosis) were restored. These results suggest that the bi-directional differentiation of GOTO cell lines stably expressing the recombinant human PPCA gene could be a model system for analyzing the functions of PPCA in peripheral neuronal cells and Schwannian cells as well as the recombinant PPCA could be a useful source for enzyme replacement therapy (ERT) for galactosialidosis patients.     

Chaperone-mediated gene therapy with recombinant AAV-PPCA in a new mouse model of type I sialidosis

The lysosomal storage disease sialidosis is caused by a primary deficiency of the sialidase N-acetyl-α-neuraminidase-1 (NEU1). Patients with type I sialidosis develop an attenuated, non-neuropathic form of the disease also named cherry red spot myoclonus syndrome, with symptoms arising during juvenile/ adult age. NEU1 requires binding to its chaperone, protective protein/cathepsin A (PPCA), for lysosomal compartmentalization, stability and catalytic activation. We have generated a new mouse model of type I sialidosis that ubiquitously expresses a NEU1 variant carrying a V54M amino acid substitution identified in an adult patient with type I sialidosis. Mutant mice developed signs of lysosomal disease after 1 year of age, predominantly in the kidney, albeit low residual NEU1 activity was detected in most organs and cell types. We demonstrate that the activity of the mutant enzyme could be effectively increased in all systemic tissues by chaperone-mediated gene therapy with a liver-tropic recombinant AAV2/8 vector expressing PPCA. This resulted in clear amelioration of the disease phenotype. These results suggest that at least some of the NEU1 mutations associated with type I sialidosis may respond to PPCA-chaperone-mediated gene therapy.     

Identification of a point mutation in the human lysosomal alpha-glucosidase gene causing infantile glycogenosis type II

Two patients in a consanguineous Indian family with infantile glycogenosis type II were found to have a G to A transition in exon 11 of the human lysosomal alpha-glucosidase gene. Both patients were homozygous and both parents were heterozygous for the mutant allele. The mutation causes a Glu to Lys substitution at amino acid position 521, just three amino acids downstream from the catalytic site at Asp-518. The mutation was introduced in wild type lysosomal alpha-glucosidase cDNA and the mutant construct was expressed in vitro and in vivo. The Glu to Lys substitution is proven to account for the abnormal physical properties of the patients lysosomal alpha-glucosidase precursor and to prevent the formation of catalytically active enzyme. In homozygous form it leads to the severe infantile phenotype of glycogenosis type II.     

A mutation (Pro-30 to Leu) in CYP21 represents a potential nonclassic steroid 21-hydroxylase deficiency allele

The mild nonclassic form of steroid 21-hydroxylase deficiency is one of the most common autosomal recessive disorders in humans, occurring in almost 1% of caucasians and about 3% of Ashkenazi Jews. Many patients with this disorder carry a Val-281----Leu missense mutation in the CYP21 gene. This and most other mutations causing 21-hydroxylase deficiency are normally present in the CYP21P pseudogene and have presumably been transferred to CYP21 by gene conversion. To identify other potential nonclassic alleles, we used recombinant vaccinia virus to express two mutant enzymes carrying the mutations Pro-30----Leu (normally present in CYP21P) and Ser-268----Thr (considered a normal polymorphism of CYP21). Whereas the activity of the protein carrying the Ser----Thr mutation was indeed indistinguishable from the wild type, the enzyme with the Pro----Leu substitution had 60% of wild-type activity for 17-hydroxyprogesterone and about 30% of normal activity for progesterone when assayed in intact cells. When kinetic analysis of the latter mutant enzyme was performed in cellular lysates, the first order rate constants (maximum velocity/dissociation constant) for both substrates were reduced 10- to 20-fold compared with those for the wild-type enzyme. Pro-30 is conserved in many microsomal P450 enzymes and may be important for proper orientation of the enzyme with respect to the aminoterminal transmembrane segment. The Pro----Leu mutation was present in 5 of 18 patients with nonclassic 21-hydroxylase deficiency, suggesting that this mutation indeed acts as a nonclassic deficiency allele.     

The relation between human lysosomal beta-galactosidase and its protective protein

Cultured skin fibroblasts from patients with the lysosomal storage disease galactosialidosis lack a 54-kDa protein which is a precursor of 32-kDa and 20-kDa proteins, which immunoprecipitate with human anti-beta-galactosidase antiserum. The lack of a 32-kDa "protective protein" results in a combined deficiency of beta-galactosidase and sialidase. The mechanism of protection of lysosomal beta-galactosidase against proteolytic degradation is elucidated by sucrose density gradient centrifugation and immunoprecipitation studies. In normal fibroblasts at the low intralysosomal pH, more than 85% of beta-galactosidase exists as a high molecular weight (600-700 kDa) multimer and about 10% as a monomer of 64-kDa. In mutant cells from galactosialidosis patients, the residual enzyme activity, about 10%, is present as a monomer and no multimer exists. After addition of the 54-kDa precursor form of the protective protein, the density pattern of beta-galactosidase in galactosialidosis cells is normalized. Immunoprecipitation studies after sucrose density gradient centrifugation on homogenate and on purified beta-galactosidase from normal fibroblasts show that the protective protein is associated only with the multimeric form of beta-galactosidase. We propose that intralysosomal protection against proteolysis of beta-galactosidase and sialidase is accomplished by aggregation into a high molecular weight complex consisting of multimeric beta-galactosidase, sialidase, and protective protein. The genetic deficiency of the latter, as in galactosialidosis, results in a rapid degradation of monomeric beta-galactosidase and a loss of sialidase activity.     

Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). An intermediate clinical phenotype caused by substitution of valine for glycine at position 137 of arylsulfatase B.

The Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI) is a lysosomal storage disease with autosomal recessive inheritance caused by deficiency of the enzyme arylsulfatase B. Severe, intermediate, and mild forms of the disease have been described. The molecular correlate of the clinical heterogeneity is not known at present. To identify the molecular defect in a patient with the intermediate form of the disease, arylsulfatase B mRNA from his fibroblasts was reverse-transcribed, amplified by the polymerase chain reaction, and subcloned. Three point mutations were detected by DNA sequence analysis, two of which, a silent A to G transition at nucleotide 1191 and a G to A transition at nucleotide 1126 resulting in a methionine for valine 376 substitution, were polymorphisms. A G to T transversion at nucleotide 410 causing a valine for glycine 137 substitution (G137V) was identified as the mutation underlying the Maroteaux-Lamy phenotype of the patient, who was homozygous for the allele. The kinetic parameters of the mutant arylsulfatase B enzyme toward a radiolabeled trisaccharide substrate were normal excluding an alteration of the active site. The G137V mutation did not affect the synthesis but severely reduced the stability of the arylsulfatase B precursor. While the wild type precursor is converted by limited proteolysis in late endosomes or lysosomes to a mature form, the majority of the mutant precursor was degraded presumably in a compartment proximal to the trans Golgi network and only a small amount escaped to the lysosomes accounting for the low residual enzyme activity in fibroblasts of a patient with the juvenile form of the disease.     

Mutator phenotype of MUTYH-null mouse embryonic stem cells

To evaluate the antimutagenic role of a mammalian mutY homolog, namely the Mutyh gene, which encodes adenine DNA glycosylase excising adenine misincorporated opposite 8-oxoguanine in the template DNA, we generated MUTYH-null mouse embryonic stem (ES) cells. In the MUTYH-null cells carrying no adenine DNA glycosylase activity, the spontaneous mutation rate increased 2-fold in comparison with wild type cells. The expression of wild type mMUTYH or mutant mMUTYH protein with amino acid substitutions at the proliferating cell nuclear antigen binding motif restored the increased spontaneous mutation rates of the MUTYH-null ES cells to the wild type level. The expression of a mutant mMUTYH protein with an amino acid substitution (G365D) that corresponds to a germ-line mutation (G382D) found in patients with multiple colorectal adenomas could not suppress the elevated spontaneous mutation rate of the MUTYH-null ES cells. Although the recombinant mMUTYH(G365D) purified from Escherichia coli cells had a substantial level of adenine DNA glycosylase activity as did wild type MUTYH, no adenine DNA glycosylase activity was detected in the MUTYH-null ES cells expressing the mMUTYH(G365D) mutant protein. The germ-line mutation (G382D) of the human MUTYH gene is therefore likely to be responsible for the occurrence of a mutator phenotype in these patients.     

Mannose 6-phosphate-independent targeting of lysosomal enzymes in I- cell disease B lymphoblasts

B lymphocytes from patients with I-cell disease (ICD) maintain normal cellular levels of lysosomal enzymes despite a deficiency of the enzyme UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1- phosphotransferase. We find that an ICD B lymphoblastoid cell line targets about 45% of the lysosomal protease cathepsin D to dense lysosomes. This targeting occurs in the absence of detectable mannose 6- phosphate residues on the cathepsin D and is not observed in ICD fibroblasts. The secretory protein pepsinogen, which is closely related to cathepsin D in both amino acid sequence and three-dimensional structure, is mostly excluded from dense lysosomes, indicating that the lymphoblast targeting pathway is specific. Carbohydrate residues are not required for lysosomal targeting, since a non-glycosylated mutant cathepsin D is sorted with comparable efficiency to the wild type protein. Analysis of a number of cathepsin D/pepsinogen chimeric proteins indicates that an extensive polypeptide determinant in the cathepsin D carboxyl lobe can confer efficient lysosomal sorting when introduced into the pepsinogen sequence. This determinant overlaps but is not identical to the recognition marker for phosphotransferase. These results indicate that a specific protein recognition event underlies Man-6-P-independent lysosomal sorting in ICD lymphoblasts.     

The acylated precursor form of the colicin A lysis protein is a natural substrate of the DegP protease.

The acylated precursor form of the colicin A lysis protein (pCalm) is specifically cleaved by the DegP protease into two acylated fragments of 6 and 4.5 kilodaltons (kDa). This cleavage was observed after globomycin treatment, which inhibits the processing of pCalm into mature colicin A lysis protein (Cal) and the signal peptide. The cleavage took place in lpp, pldA, and wild-type strans carrying plasmids which express the lysis protein following SOS induction and also in cells containing a plasmid which expresses it under the control of the tac promoter. Furthermore, the DegP protease was responsible for the production of two acylated Cal fragments of 3 and 2.5 kDa in cells carrying plasmids which overproduce the Cal protein, without treatment with globomycin. DegP could also cleave the acylated precursor form of a mutant Cal protein containing a substitution in he amino-terminal portion of the protein, but not that of a mutant Cal containing a frameshift mutation in its carboxyl-terminal end. The functions of Cal in causing protein release, quasi-lysis, and lethality were increased in degP41 cells, suggesting that mature Cal was produced in higher amounts in the mutant than in the wild type. These effects were limited in cells deficient in phospholipase A. Interactions between the DegP protease and phospholipase A were suggested by the characteristics of degP pldA double mutants.     

Processing of lysosomal beta-galactosidase. The C-terminal precursor fragment is an essential domain of the mature enzyme

Lysosomal beta-D-galactosidase (beta-gal), the enzyme deficient in the autosomal recessive disorders G(M1) gangliosidosis and Morquio B, is synthesized as an 85-kDa precursor that is C-terminally processed into a 64-66-kDa mature form. The released approximately 20-kDa proteolytic fragment was thought to be degraded. We now present evidence that it remains associated to the 64-kDa chain after partial proteolysis of the precursor. This polypeptide was found to copurify with beta-gal and protective protein/cathepsin A from mouse liver and Madin-Darby bovine kidney cells and was immunoprecipitated from human fibroblasts but not from fibroblasts of a G(M1) gangliosidosis and a galactosialidosis patient. Uptake of wild-type protective protein/cathepsin A by galactosialidosis fibroblasts resulted in a significant increase of mature and active beta-gal and its C-terminal fragment. Expression in COS-1 cells of mutant cDNAs encoding either the N-terminal or the C-terminal domain of beta-gal resulted in the synthesis of correctly sized polypeptides without catalytic activity. Only when co-expressed, the two subunits associate and become catalytically active. Our results suggest that the C terminus of beta-gal is an essential domain of the catalytically active enzyme and provide evidence that lysosomal beta-galactosidase is a two-subunit molecule. These data may give new significance to mutations in G(M1) gangliosidosis patients found in the C-terminal part of the molecule.     

Mitochondrial import and processing of wild type and type III mutant isovaleryl-CoA dehydrogenase

Isovaleric acidemia is a rare inborn error of metabolism caused by a deficiency of isovaleryl-CoA dehydrogenase (IVD), a nucleus-encoded, homotetrameric, mitochondrial flavoenzyme that catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA. We have previously identified a nucleotide deletion in the gene for IVD in fibroblasts from a patient with isovaleric acidemia leading to a shift in reading frame and premature termination of translation. The mutant IVD precursor is imported and processed to mature size, but no active enzyme is detected in mutant fibroblasts or expressed in Escherichia coli. Examination of the crystal structure of human IVD reveals that the C terminus is involved in tetramer stability. In vitro mitochondrial import experiments show that wild type IVD protein rapidly and stably forms mature homotetramer following import, whereas Type III mutant protein never forms stable oligomers. An additional series of mutant proteins with truncations and/or alterations in the C-terminal sequence implicates the C terminus of IVD in both enzyme activity and tetramer stability. Importantly, a dimeric intermediate in the folding pathway for wild type IVD has been identified in the in vitro mitochondrial import experiments, the first report of such an intermediate in the biogenesis of an acyl-CoA dehydrogenase.     

A single point mutation in the low-density lipoprotein receptor switches the degradation of its mature protein from the proteasome to the lysosome

Pathogenic mutations in the low-density lipoprotein receptor prevent cholesterol uptake and cause familial hypercholesterolemia. In comparison to the biogenesis and endocytic trafficking of this receptor and some of its mutants, their degradation mechanisms are not well understood. Therefore, to gain some insights into this aspect, we analyzed the effects of proteasomal and lysosomal inhibitors on the levels of the wild type low-density lipoprotein receptor and a mutant form, C358Y, which was prevalent in a sample of Spanish familial hypercholesterolemia patients. In transfected cells, the mutant C358Y exhibited lower activity than the wild type receptor, as well as retarded post-translational processing of its precursor to the mature form. Interestingly, about 30% of the mutant precursor was degraded by a lysosomal pathway. Moreover, its mature form was more rapidly degraded than the wild type receptor (half lives of 5.3 and 10.9 h, respectively) and its degradation was exclusively dependent on a lysosomal pathway. In contrast, the mature form of the wild type receptor was mainly degraded by proteasomes and, to a minor extent (30%), by lysosomes. We conclude that a single mutation in the low-density lipoprotein receptor switches the degradation of the mature receptor from a proteasomal to a lysosomal pathway which degrades the protein at a faster rate. This suggests cooperation of proteasomes and lysosomes in the degradation of the low-density lipoprotein receptor and adds an intriguing new aspect to our understanding of receptor-mediated endocytosis.     

Dominant negative mutants of ornithine decarboxylase.

Conserved lysines of mouse ornithine decarboxylase were individually mutated to arginines. The mutations at amino acid residues 69, 115, and 169 greatly reduced or abolished enzymatic activity. Lysine 69 is the site of Schiff base formation with the cofactor pyridoxal phosphate; the functional role of the other two lysines essential for activity is not known. Coexpression of wild type ornithine decarboxylase along with the lysine 115 to arginine mutant reduced the activity of the former without diminishing the amount of wild type protein. This form of negative complementation was seen when wild type and mutant protein were coexpressed either by in vitro translation or in bacteria. The data are consistent with the conclusion that a wild type and mutant subunit form a heterodimer that is enzymatically inactive.     

Wild type and mutant amyloid precursor proteins influence downstream effects of proteasome and autophagy inhibition

Cells rely on complementary proteolytic pathways including the ubiquitin–proteasome system and autophagy to maintain proper protein degradation. There is known to be considerable interplay between them, whereby the loss of one clearance system results in compensatory changes in other proteolytic pathways of the cell. Disturbances in proteolysis are known to occur in Alzheimer's disease, and potentially contribute to neurophysiological and neurodegenerative processes. Currently, few data are available on how the presence of wild type and mutant amyloid precursor protein (APPwt and APPmut) potentially alters the reciprocal interplay between the different intracellular proteolytic pathways. This study used human SH-SY5Y neuronal cell lines, and SH-SY5Y transfected with either APPwt or APPmut (valine-to-glycine substitution at position 717), in order to explore if the presence of APPwt or APPmut altered the downstream effects of pharmacological proteasome or autophagy inhibition. The occurrence of APPwt or APPmut was observed to disturb proteasome or autophagy activities upon treatment with proteasome inhibitors or authophagy inhibitors. Interestingly, APPwt and APPmut expression was observed to significantly and robustly enhance the induction in cathepsin B following the administration of an established proteasome inhibitor. The presence of APPwt and APPmut also significantly reduced the elevation in ubiquitinated proteins following proteasome inhibitor treatments. Our data strongly suggest that APP is able to affect the downstream effects of protease inhibition in neural cells including enhancement of cathepsin B activity, with these changes in cathepsin B significantly and inversely related to the levels of ubiquitinated protein.     

Molecular basis of aberrant apical protein transport in an intestinal enzyme disorder

The impaired sorting profile to the apical membrane of human intestinal sucrase-isomaltase is the underlying cause in the pathogenesis of a novel phenotype of intestinal congenital sucrase-isomaltase deficiency. Molecular characterization of this novel phenotype reveals a point mutation in the coding region of the sucrase-isomaltase (SI) gene that results in an amino acid substitution of a glutamine by arginine at residue 117 of the isomaltase subunit. This substitution is located in a domain revealing features of a trefoil motif or a P-domain in immediate vicinity of the heavily O-glycosylated stalk domain. Expression of the mutant SI phenotype in epithelial Madin-Darby canine kidney cells reveals a randomly targeted SI protein to the apical and basolateral membranes confirming an exclusive role of the Q117R mutation in generating this phenotype. Unlike wild type SI, the mutant protein is completely extractable with Triton X-100 despite the presence of O-glycans that serve in the wild type protein as an apical sorting signal and are required for the association of SI with detergent-insoluble lipid microdomains. Obviously the O-glycans are not adequately recognized in the context of the mutant SI, most likely due to altered folding of the P-domain that ultimately affects the access of the O-glycans to a putative sorting element.