Human alpha-Gal Research Articles

Catalytic Mechanism of Human α-Galactosidase

The enzyme alpha-galactosidase (alpha-GAL, also known as alpha-GAL A; E.C. 3.2.1.22) is responsible for the breakdown of alpha-galactosides in the lysosome. Defects in human alpha-GAL lead to the development of Fabry disease, a lysosomal storage disorder characterized by the buildup of alpha-galactosylated substrates in the tissues. alpha-GAL is an active target of clinical research: there are currently two treatment options for Fabry disease, recombinant enzyme replacement therapy (approved in the United States in 2003) and pharmacological chaperone therapy (currently in clinical trials). Previously, we have reported the structure of human alpha-GAL, which revealed the overall structure of the enzyme and established the locations of hundreds of mutations that lead to the development of Fabry disease. Here, we describe the catalytic mechanism of the enzyme derived from x-ray crystal structures of each of the four stages of the double displacement reaction mechanism. Use of a difluoro-alpha-galactopyranoside allowed trapping of a covalent intermediate. The ensemble of structures reveals distortion of the ligand into a (1)S(3) skew (or twist) boat conformation in the middle of the reaction cycle. The high resolution structures of each step in the catalytic cycle will allow for improved drug design efforts on alpha-GAL and other glycoside hydrolase family 27 enzymes by developing ligands that specifically target different states of the catalytic cycle. Additionally, the structures revealed a second ligand-binding site suitable for targeting by novel pharmacological chaperones.

The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency in alpha-galactosidase A (alpha-Gal A) activity and subsequent accumulation of the substrate globotriaosylceramide (GL-3), which contributes to disease pathology. The pharmacological chaperone (PC) DGJ (1-deoxygalactonojirimycin) binds and stabilizes alpha-Gal A, increasing enzyme levels in cultured cells and in vivo. The ability of DGJ to reduce GL-3 in vivo was investigated using transgenic (Tg) mice that express a mutant form of human alpha-Gal A (R301Q) on a knockout background (Tg/KO), which leads to GL-3 accumulation in disease-relevant tissues. Four-week daily oral administration of DGJ to Tg/KO mice resulted in significant and dose-dependent increases in alpha-Gal A activity, with concomitant GL-3 reduction in skin, heart, kidney, brain, and plasma; 24-week administration resulted in even greater reductions. Compared to daily administration, less frequent DGJ administration, including repeated cycles of 4 days with DGJ followed by 3 days without or every other day with DGJ, resulted in even greater GL-3 reductions that were comparable to those obtained with Fabrazyme. Collectively, these data indicate that oral administration of DGJ increases mutant alpha-Gal A activity and reduces GL-3 in disease-relevant tissues in Tg/KO mice, and thus merits further evaluation as a treatment for Fabry disease.

Naked Plasmid DNA-Based α-Galactosidase A Gene Transfer Partially Reduces Systemic Accumulation of Globotriaosylceramide in Fabry Mice

Fabry disease is an X-linked recessive inborn metabolic disorder in which a deficiency in lysosomal enzyme alpha-galactosidase A (Gal A) causes the systemic accumulation of globotriaosylceramide (Gb3). Although many investigators have attempted to treat alpha-Gal A knock-out mice (Fabry mice) with gene therapy, no report has demonstrated therapeutic effects by the retrograde renal vein injection of naked DNA. We recently developed a naked plasmid vector-mediated kidney-targeted gene transfer technique. A solution containing naked plasmid DNA encoding human alpha-Gal A (pKSCX-alpha-Gal A) was rapidly injected into the left kidney of Fabry mice (pKSCX-alpha-Gal A mice). pKSCX was used for mock transfections (pKSCX mice). We confirmed that vector-derived human alpha-Gal A mRNA was present in the left kidney but not in other tissues, by reverse transcriptase polymerase chain reaction. Compared with the pKSCX mice, the pKSCX-alpha-Gal A mice showed partial therapeutic effects: increased alpha-Gal A activity in the injected kidney and in the liver, heart, and plasma, and decreased Gb3 in the injected kidney, contralateral kidney, liver, heart, and spleen. Our results demonstrated that, although further studies are needed to improve the outcome, this method has promise as a potential treatment option for Fabry disease.

Caveolin-associated Accumulation of Globotriaosylceramide in the Vascular Endothelium of α-Galactosidase A Null Mice

Cardiovascular complications, including stroke and myocardial infarction, result in premature mortality in patients with Fabry disease, an X-linked deficiency of alpha-galactosidase A (alpha-Gal A). The enzymatic defect results in the deposition of globotriaosylceramide (Gb3) in the vascular endothelium. To better understand the underlying pathogenesis of Fabry disease, the caveolar lipid content of primary cultured mouse aortic endothelial cells isolated from alpha-Gal A null mice was measured. Lipid mass analysis revealed that the excessive Gb3 in cultured alpha-Gal A-deficient mouse aortic endothelial cells accumulated in endothelial plasma membrane caveolar fractions. The levels of glucosylceramide and lactosylceramide increased in parallel with Gb3 levels in an age-dependent manner, whereas globotetraosylceramide (Gb4) levels reached maximal levels by 6 months of age and then rapidly decreased at older ages. The levels of cholesterol enriched in caveolar membranes declined in parallel with the progressive deposition of Gb3. Depleting Gb3 with recombinant human alpha-Gal A protein or d-threo-ethylenedioxyphenyl-P4, an inhibitor of glucosylceramide synthase, restored cholesterol in cultured alpha-Gal A-deficient mouse aortic endothelial cell caveolae. By contrast, recombinant human alpha-Gal A was less effective in normalizing the cholesterol content. These results demonstrate the caveolar accumulation of glycosphingolipids in an in vitro model of a lysosomal storage disease and raise the possibility that dynamic changes in the composition of plasma membrane lipid microdomains may mediate the endothelial dysfunction seen in Fabry disease.

Efficient correction of Fabry mice and patient cells mediated by lentiviral transduction of hematopoietic stem/progenitor cells

A deficiency in alpha-galactosidase A (alpha-gal A) activity causes Fabry disease. Virus-based delivery of genes can correct cells and establish a sustained supply of therapeutic proteins. Recombinant lentiviral vectors (LVs) show promise in this context. We first demonstrate LV-mediated marking of peripheral blood (PB) cells by transduction/transplantation of hematopoietic stem/progenitor cells. Stable enGFP expression was observed in PB for 37 weeks. Next, we transplanted Fabry mice with bone marrow mononuclear cells (BMMNCs) transduced a single time with a LV encoding the human alpha-gal A cDNA. Sustained expression of functional alpha-gal A in Fabry mice was observed over 24 weeks. Plasma alpha-gal A activity from treated Fabry mice was two-fold higher than wild-type controls. Increased alpha-gal A activity, often to supra-normal levels, and reduction of globotriaosylceramide, a glycolipid that accumulates in Fabry disease, was observed in all organs assessed. In secondary bone marrow transplantations, Fabry mice showed multilineage marking of PB, splenocytes and BMMNCs, along with therapeutic levels of alpha-gal A activity in plasma and organs over 20 weeks. Lastly, we transduced mobilized PB CD34(+) cells from a Fabry patient and observed corresponding enzymatic increases. Thus a single LV-mediated transduction of primitive hematopoietic cells can result in sustained correction for Fabry disease.

Fabry disease: characterization of alpha-galactosidase A double mutations and the D313Y plasma enzyme pseudodeficiency allele.

Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the gene encoding the lysosomal exoglycohydrolase, alpha-galactosidase A (alpha-Gal A; GLA). In two unrelated classically affected males, two alpha-Gal A missense mutations were identified: R112C + D313Y (c.334C>T + c.937G>T) and C172G + D313Y (c.514T>G + c.937G>T). The D313Y lesion was previously identified in classically affected males as the single mutation [Eng et al., 1993] or in cis with another missense mutation, D313Y + G411D (c.937G>T + c.1232G>A) [Guffon et al., 1998]. To determine whether the D313Y mutation was a deleterious mutation or a coding region sequence variant, the frequency of D313Y in normal X-chromosomes, as well as its enzymatic activity and subcellular localization in COS-7 cells was determined. D313Y occurred in 0.45% of 883 normal X-chromosomes, while the R112C, C172G, and G411D missense mutations were not detected in over 500 normal X-chromosomes. Expression of D313Y in COS-7 cells resulted in approximately 60% of wild-type enzymatic activity and showed lysosomal localization, while R112C, C172G, G411D, and the double-mutated constructs had markedly reduced or no detectable activity and were all retained in the endoplasmic reticulum. The expressed D313Y enzyme was stable at lysosomal pH (pH 4.6), while at neutral pH (pH 7.4), it had decreased activity. A molecular homology model of human alpha-Gal A, based on the X-ray crystal structure of chicken alpha-galactosidase B (alpha-Gal B; alpha-N-acetylgalactosaminidase) was generated [Garman et al., 2002], which provided evidence that D313Y did not markedly disrupt the alpha-Gal A enzyme structure. Thus, D313Y is a rare exonic variant with about 60% of wild-type activity in vitro and reduced activity at neutral pH, resulting in low plasma alpha-Gal A activity.

Long-term correction of globotriaosylceramide storage in Fabry mice by recombinant adeno-associated virus-mediated gene transfer.

Fabry disease is an X-linked recessive inborn metabolic disorder characterized by systemic and vascular accumulation of globotriaosylceramide (Gb(3)) caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). The condition is associated with an increased morbidity and mortality due to renal failure, cardiac disease, and early onset of stroke. Hemizygous males are primarily affected clinically with variable expression in heterozygous females. Gene-therapy trials have been initiated recently in alpha-gal A knockout mouse models of Fabry disease by using a variety of viral vectors. In the present investigation we administered single i.v. injections of 1 x 10(10) genomes of recombinant adeno-associated virus (rAAV) encoding the human alpha-gal A gene driven by a modified chicken beta-actin (CAG) promoter to alpha-gal A knockout (Fabry) mice. Transgenic mice were analyzed for expression of alpha-gal A activity and Gb(3) levels in liver, kidney, heart, spleen, small intestine, lung, and brain. Administration of the rAAV-CAG-hAGA vector resulted in stable expression of alpha-gal A in organs of the Fabry mice for >6 months. alpha-Gal A activity in the organs became equal to or higher than that of wild-type mice. Accumulated Gb(3) in the liver, heart, and spleen was reduced to that of wild-type mice with lesser but significant reductions in kidney, lung, and small intestine. Injection of the rAAV-CAG-hAGA construct into skeletal muscle did not result in expression of alpha-gal A in it or in other tissues. This study provides a basis for a simple and efficient gene-therapy approach for patients with Fabry disease and is indicative of its potential for the treatment of other lysosomal storage disorders.

Recombinant Enzyme Therapy for Fabry Disease: Absence of Editing of Human α-Galactosidase A mRNA

For more than a decade, protein-replacement therapy has been employed successfully for the treatment of Gaucher disease. Recently, a comparable therapy has become available for the related lipid-storage disorder Fabry disease. Two differently produced recombinant alpha-galactosidase A (alpha-gal A) preparations are used independently for this purpose. Agalsidase alpha is obtained from human fibroblasts that have been modified by gene activation; agalsidase beta is obtained from Chinese hamster ovary cells that are transduced with human alpha-gal A cDNA. It has previously been claimed that alpha-gal A mRNA undergoes editing, which may result in coproduction of an edited protein (Phe 396 Tyr) that might have a relevant physiological function. We therefore analyzed the occurrence of alpha-gal A editing, as well as the precise nature, in this respect, of the therapeutic enzymes. No indications were obtained for the existence of editing at the protein or RNA level. Both recombinant enzymes used in therapy are unedited and are capable of functionally correcting cultured fibroblasts from Fabry patients in their excessive globotriaosylceramide accumulation. Although RNA editing is apparently not relevant in the case of alpha-gal A, a thorough analysis of the potential occurrence of editing of transcripts is nevertheless advisable in connection with newly developed protein-replacement therapies.

Enzyme replacement and beyond

During the last decade, enzyme replacement therapy for lysosomal storage diseases became a reality with the demonstration of its safety and effectiveness in type 1 Gaucher disease. Currently, enzyme replacement and several other potential therapeutic strategies are being developed for selected lysosomal storage diseases, including Fabry disease due to the deficient activity of alpha-galactosidase A (alpha-Gal A). The development and clinical evaluation of these new therapies require a stepwise process, each step being rigorously reviewed and approved by national or international regulatory agencies. For lethal disorders that affect small populations, such as many inherited metabolic diseases, this process can be accelerated by 'orphan drug' and 'fast track' regulations. As an example of the drug development process, the development of recombinant human alpha-Gal A (r-halphaGal A) replacement for Fabry disease is presented, including the preclinical studies in the 'Fabry mouse' model, and the clinical phase 1/2, phase 3, and phase 3 extension studies, which demonstrate the safety and efficacy of this new therapy.

A Phase 1/2 Clinical Trial of Enzyme Replacement in Fabry Disease: Pharmacokinetic, Substrate Clearance, and Safety Studies

Fabry disease results from deficient alpha-galactosidase A (alpha-Gal A) activity and the pathologic accumulation of the globotriaosylceramide (GL-3) and related glycosphingolipids, primarily in vascular endothelial lysosomes. Treatment is currently palliative, and affected patients generally die in their 40s or 50s. Preclinical studies of recombinant human alpha-Gal A (r-halphaGalA) infusions in knockout mice demonstrated reduction of GL-3 in tissues and plasma, providing rationale for a phase 1/2 clinical trial. Here, we report a single-center, open-label, dose-ranging study of r-halphaGalA treatment in 15 patients, each of whom received five infusions at one of five dose regimens. Intravenously administered r-halphaGalA was cleared from the circulation in a dose-dependent manner, via both saturable and non-saturable pathways. Rapid and marked reductions in plasma and tissue GL-3 were observed biochemically, histologically, and/or ultrastructurally. Clearance of plasma GL-3 was dose-dependent. In patients with pre- and posttreatment biopsies, mean GL-3 content decreased 84% in liver (n=13), was markedly reduced in kidney in four of five patients, and after five doses was modestly lowered in the endomyocardium of four of seven patients. GL-3 deposits were cleared to near normal or were markedly reduced in the vascular endothelium of liver, skin, heart, and kidney, on the basis of light- and electron-microscopic evaluation. In addition, patients reported less pain, increased ability to sweat, and improved quality-of-life measures. Infusions were well tolerated; four patients experienced mild-to-moderate reactions, suggestive of hypersensitivity, that were managed conservatively. Of 15 patients, 8 (53%) developed IgG antibodies to r-halphaGalA; however, the antibodies were not neutralizing, as indicated by unchanged pharmacokinetic values for infusions 1 and 5. This study provides the basis for a phase 3 trial of enzyme-replacement therapy for Fabry disease.

Fabry Disease: Preclinical Studies Demonstrate the Effectiveness of α-Galactosidase A Replacement in Enzyme-Deficient Mice

Preclinical studies of enzyme-replacement therapy for Fabry disease (deficient alpha-galactosidase A [alpha-Gal A] activity) were performed in alpha-Gal A-deficient mice. The pharmacokinetics and biodistributions were determined for four recombinant human alpha-Gal A glycoforms, which differed in sialic acid and mannose-6-phosphate content. The plasma half-lives of the glycoforms were approximately 2-5 min, with the more sialylated glycoforms circulating longer. After intravenous doses of 1 or 10 mg/kg body weight were administered, each glycoform was primarily recovered in the liver, with detectable activity in other tissues but not in the brain. Normal or greater activity levels were reconstituted in various tissues after repeated doses (10 mg/kg every other day for eight doses) of the highly sialylated AGA-1 glycoform; 4 d later, enzyme activity was retained in the liver and spleen at levels that were, respectively, 30% and 10% of that recovered 1 h postinjection. Importantly, the globotriaosylceramide (GL-3) substrate was depleted in various tissues and plasma in a dose-dependent manner. A single or repeated doses (every 48 h for eight doses) of AGA-1 at 0.3-10.0 mg/kg cleared hepatic GL-3, whereas higher doses were required for depletion of GL-3 in other tissues. After a single dose of 3 mg/kg, hepatic GL-3 was cleared for > or =4 wk, whereas cardiac and splenic GL-3 reaccumulated at 3 wk to approximately 30% and approximately 10% of pretreatment levels, respectively. Ultrastructural studies demonstrated reduced GL-3 storage posttreatment. These preclinical animal studies demonstrate the dose-dependent clearance of tissue and plasma GL-3 by administered alpha-Gal A, thereby providing the in vivo rationale-and the critical pharmacokinetic and pharmacodynamic data-for the design of enzyme-replacement trials in patients with Fabry disease.

Circulating alpha-galactosidase A derived from transduced bone marrow cells: relevance for corrective gene transfer for Fabry disease.

Fabry disease is caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). We previously engineered a retrovirus encoding human alpha-gal A and demonstrated enzymatic correction of patient cells. Further, we demonstrated metabolic cooperativity, in that corrected cells secrete alpha-gal A that can be taken up and utilized by bystander cells in vitro. In the present study, we created a system to examine and quantitate this phenomenon in vivo. To differentiate from endogenous alpha-gal A, we constructed a retroviral vector (pUMFG/alpha-gal A/FLAG) containing a fusion form of alpha-gal A with a specific tag sequence at the carboxy terminus. The catalytic activity of the fusion protein was identical to wild-type alpha-gal A. The fusion protein was overexpressed in and secreted by transduced patient cells. In uptake studies, the fusion protein was detected in the lysosome-enriched fraction of recipient cells. We then examined the effectiveness of the pUMFG/alpha-g A/FLAG retroviral vector in vivo. Murine bone marrow (BM) cells were transduced and transplanted into irradiated hosts. After 9 weeks, proviral DNA was detected by PCR in peripheral blood and BM mononuclear cells. More importantly, specific fusion protein enzymatic activity could be demonstrated in those cells and in plasma. Thus, we have demonstrated that overexpressed alpha-gal A enters the circulation from transduced BM cells and is stable over a significant period of time.

Human alpha-galactosidase A: characterization of the N-linked oligosaccharides on the intracellular and secreted glycoforms overexpressed by Chinese hamster ovary cells.

Human alpha-galactosidase A (alpha-Gal A) is the lysosomal glycohydrolase that cleaves the terminal alpha-galactosyl moieties of various glycoconjugates. Overexpression of the enzyme in Chinese hamster ovary (CHO) cells results in high intracellular enzyme accumulation and the selective secretion of active enzyme. Structural analysis of the N -linked oligosaccharides of the intracellular and secreted glycoforms revealed that the secreted enzyme's oligosaccharides were remarkably heterogeneous, having high mannose (63%), complex (30%), and hybrid (5%) structures. The major high mannose oligosaccharides were Man5-7GlcNAc2 species. Approximately 40% of the high mannose and 30% of the hybrid oligosaccharides had phosphate monoester groups. The complex oligosaccharides were mono-, bi-, 2,4-tri-, 2,6-tri- and tetraantennary with or without core-region fucose, many of which had incomplete outer chains. Approximately 30% of the complex oligosaccharides were mono- or disialylated. Sialic acids were mostly N -acetylneuraminic acid and occurred exclusively in alpha2, 3-linkage. In contrast, the intracellular enzyme had only small amounts of complex chains (7.7%) and had predominantly high mannose oligosaccharides (92%), mostly Man5GlcNAc2 and smaller species, of which only 3% were phosphorylated. The complex oligosaccharides were fucosylated and had the same antennary structures as the secreted enzyme. Although most had mature outer chains, none were sialylated. Thus, the overexpression of human alpha-Gal A in CHO cells resulted in different oligosaccharide structures on the secreted and intracellular glycoforms, the highly heterogeneous secreted forms presumably due to the high level expression and impaired glycosylation in the trans- Golgi network, and the predominately Man5-7GlcNAc2 cellular glycoforms resulting from carbohydrate trimming in the lysosome.

Gene transfer and expression of human alpha-galactosidase from mouse muscle in vitro and in vivo.

Lysosomal storage disorders are amenable to treatment by enzyme replacement. Genetic modification of muscle via direct injection of expression vectors might represent an alternative method of providing the defective enzymes, if adequate and long-lasting expression levels can be achieved in muscle. We have used the C2C12 mouse myogenic cell line to study the effect of combination of muscle-specific regulatory elements on the expression of the human lysosomal enzyme alpha-galactosidase (alpha-gal). In differentiated myotubes, a construct containing the myosin light chain 1/3 enhancer in combination with the human cytomegalovirus promoter resulted in higher expression than constructs combining the same enhancer with the rabbit beta-myosin heavy chain promoter, or containing the CMV promoter only. Increased enzymatic activity was detectable both in cell extracts and in supernatants. Furthermore, human fibroblasts deficient in alpha-gal were able to take up the enzyme from medium conditioned by transfected myoblasts. This did not occur in the presence of mannose-6-phosphate which indicates that the uptake was via mannose-6-phosphate receptors. To our knowledge, this is the first report in which a correctly processed form of human alpha-gal was expressed and secreted from differentiated muscle cells. Direct injection of a plasmid expression vector into mouse tibialis anterior muscle showed significantly increased levels of alpha-gal 7 days after injection.

Retroviral coexpression of a multidrug resistance gene (MDR1) and human alpha-galactosidase A for gene therapy of Fabry disease.

Human alpha-galactosidase A (alpha-Gal A; EC.3.2.1.22) is a lysosomal exoglycosidase encoded by a gene on Xq22. Deficiencies of this enzyme result in Fabry disease, an X-chromosome-linked recessive disorder that leads to premature death in affected males. For treatment of genetic diseases, we have developed a retroviral vector system, pSXLC/pHa, that enables coexpression of drug-selectable markers with a second nonselectable gene as part of a bicistronic message using the promoter from the Harvey murine sarcoma virus and an internal ribosomal entry site (IRES) from encephalomyocarditis virus. Retroviral vectors based on this system that carry the human alpha-Gal A cDNA either upstream (pHa-alpha Gal-IRES-MDR) or downstream (pHa-MDR-IRES-alpha Gal) from the IRES relative to the drug-selectable MDR1 (P-glycoprotein) cDNA were constructed. Each of eight independent vincristine-resistant, pHa-alpha Gal-IRES-MDR-transfected clones and all four vincristine-resistant, pHa-alpha Gal-IRES-MDR retrovirus-transduced clones showed significantly higher activity of alpha-Gal A than the parental cells. More than 50% of the vincristine-resistant, pHa-MDR-IRES-alpha Gal-transfected clones and all four vincristine-resistant, pHa-MDR-IRES-alpha Gal retrovirus-transduced clones showed significantly higher activity of alpha-Gal A than the parental cells. In these bicistronic vectors, the cDNA whose translation was cap-dependent (upstream) was expressed at higher levels than when the same cDNA was translated in an IRES-dependent manner (downstream). These vectors may prove useful in the gene therapy of Fabry disease.

Fabry disease: isolation of a cDNA clone encoding human alpha-galactosidase A.

Fabry disease is an X-linked inborn error of metabolism resulting from the deficient activity of the lysosomal hydrolase, alpha-galactosidase A (alpha-Gal A; alpha-D-galactoside galactohydrolase, EC 3.2.1.22). To investigate the structure, organization, and expression of alpha-Gal A, as well as the nature of mutations in Fabry disease, a clone encoding human alpha-Gal A was isolated from a lambda gt11 human liver cDNA expression library. To facilitate screening, an improved affinity purification procedure was used to obtain sufficient homogeneous enzyme for production of monospecific antibodies and for amino-terminal and peptide microsequencing. On the basis of an amino-terminal sequence of 24 residues, two sets of oligonucleotide mixtures were synthesized corresponding to adjacent, but not overlapping, amino acid sequences. In addition, an oligonucleotide mixture was synthesized based on a sequence derived from an alpha-Gal A internal tryptic peptide isolated by reversed-phase HPLC. Four positive clones were initially identified by antibody screening of 1.4 X 10(7) plaques. Of these, only one clone (designated lambda AG18) demonstrated both antibody binding specificity by competition studies using homogeneous enzyme and specific hybridization to synthetic oligonucleotide mixtures corresponding to amino-terminal and internal amino acid sequences. Nucleotide sequencing of the 5' end of the 1250-base-pair EcoRI insert of clone lambda AG18 revealed an exact correspondence between the predicted and known amino-terminal amino acid sequence. The insert of clone lambda AG18 appears to contain the full-length coding region of the processed, enzymatically active alpha-Gal A, as well as sequences coding for five amino acids of the amino-terminal propeptide, which is posttranslationally cleaved during enzyme maturation.