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The cytoplasmic ribosomes and the rough endoplasmic reticulum (RER) produce protein in the body. The cytoplasmic ribosomes produce proteins necessary for cytoplasmic, mitochondrial, and peroxisomal functions. The RER produces the proteins required for endoplasmic, Golgi, and lysosomal functions. These proteins must be localized appropriately to carry out their intracellular and extracellular tasks. The process of directing proteins to their appropriate location is termed protein targeting.[1][2] Protein targeting may use vesicles depending on the source of the protein. Proteins from cytoplasmic ribosomes are not directed via vesicles, whereas proteins from the RER are localized in the cellular apparatus via vesicles. In protein targeting, many proteins are favorably modified by enzymes and helper proteins to improve the delivery. In the event of genetic mutations, proteins may localize inappropriately, leading to abnormal cellular function. This process's alteration can result in fatal metabolic diseases such as inclusion-cell disease (ICD).[3]

In 1972, Hickman and Neufeld [69] first provided a hypothesis for the pathophysiology of ICD. They used culture mediums to identify that I-cells could endocytose acid hydrolases put out by normal fibroblasts, but normal fibroblasts could not endocytose enzymes from I-cells. This finding led them to concentrate their theory not on the defective membranes that allowed enzymes to escape but on defective lysosomal enzymes that failed to reach their target destination.[69] Dysfunctional protein targeting may lead to the accumulation of proteins and enzymes in abnormal locations. ICD is a disorder of protein targeting. ICD, also known as Leroy I-cell disease, is an autosomal recessive lysosomal storage disorder due to a mutation of G1cNAc-1-phosphotransferase. The term 'mucolipidosis' covers diseases belonging to both mucopolysaccharidoses and sphingolipidoses.[66] ICD has features of both disease categories and is classified as such. There are 4 types of mucolipidoses: type I (sialidosis), type II (ICD), type III (pseudo-Hurler polydystrophy), and type IV (mucolipidosis). All of these diseases are classified according to the deficient or mutated enzyme.[33] Due to the similar clinical features, differentiating Hurler syndrome from ICD is a difficult task. A unique clinical feature exclusive to Hurler syndrome is a temporary acceleration of skeletal growth at 1 year of age.[70] Leroy et al first distinguished between mucopolysaccharidoses and ICD by identifying the cytoplasmic inclusions and variations in beta-glucuronidase and acid phosphatase levels. Of the 2 genes encoding the functional components of G1cNAc-1-phosphotransferase, GNPTAB has been connected with the mutations causing ICD. Mutations like frameshift, nonsense, splicing defects, missense, and deletions/duplications/insertions lead to the production of a stop codon 80% of the time. The stop codon forms an incomplete and dysfunctional enzyme or may lead to truncated enzyme subunits.[57] A majority of the mutations are frameshift mutations (39%). Kudo et al found the specific mutations of GNPTAB to be of FS211X (type 1), FS288X, FS546X, FS588X, FS737X, FS1081X, and FS1172X. In particular, FS1085X (type 1) had a frameshift mutation due to inappropriate splicing, removing exon 17. K4Q and S15Y mutations in the alpha-subunit can decrease G1cNAc-1-phosphotransferase activity in the Golgi complex.[71]

The term 'mucolipidosis' covers diseases belonging to both mucopolysaccharidoses and sphingolipidoses.[66] ICD has features of both disease categories and is classified as such. There are 4 types of mucolipidoses: type I (sialidosis), type II (ICD), type III (pseudo-Hurler polydystrophy), and type IV (mucolipidosis). All of these diseases are classified according to the deficient or mutated enzyme.[33] Due to the similar clinical features, differentiating Hurler syndrome from ICD is a difficult task. A unique clinical feature exclusive to Hurler syndrome is a temporary acceleration of skeletal growth at 1 year of age.[70] Leroy et al first distinguished between mucopolysaccharidoses and ICD by identifying the cytoplasmic inclusions and variations in beta-glucuronidase and acid phosphatase levels. Of the 2 genes encoding the functional components of G1cNAc-1-phosphotransferase, GNPTAB has been connected with the mutations causing ICD. Mutations like frameshift, nonsense, splicing defects, missense, and deletions/duplications/insertions lead to the production of a stop codon 80% of the time. The stop codon forms an incomplete and dysfunctional enzyme or may lead to truncated enzyme subunits.[57] A majority of the mutations are frameshift mutations (39%). Kudo et al found the specific mutations of GNPTAB to be of FS211X (type 1), FS288X, FS546X, FS588X, FS737X, FS1081X, and FS1172X. In particular, FS1085X (type 1) had a frameshift mutation due to inappropriate splicing, removing exon 17. K4Q and S15Y mutations in the alpha-subunit can decrease G1cNAc-1-phosphotransferase activity in the Golgi complex.[71] Although the mutation variants are evenly spread throughout GNPTAB, approximately 25% of the mutations are in the 1112-bp exon 13. Clinical severity correlates with the level of enzyme activity. Absent or severely reduced GlcNAc-1-phosphotransferase activity due to strong alterations of GNPTAB leads to the severe ICD phenotype. Having residual enzyme activity (approximately 10%) due to at least 1 GNPTAB allele being active is associated with the less severe ML III alpha/beta disease.[32][56] Patients with ML III can survive into their adulthood, whereas patients with ML II often die within the first decade of life. ML III is divided into ML IIIA (pseudo-Hurler polydystrophy) and ML IIIC (variant pseudo-Hurler polydystrophy). In ML IIIA, the G1cNAc-1-phosphotransferase activity is reduced. In ML IIIC, G1cNAc-1-phosphate transfer to alpha-methylmannoside is normal, but the transfer to lysosomal substrates is reduced.[57] Measuring G1cNAc-1-phosphotransferase levels can help differentiate ML II from other mucopolysaccharidoses.

Although the mutation variants are evenly spread throughout GNPTAB, approximately 25% of the mutations are in the 1112-bp exon 13. Clinical severity correlates with the level of enzyme activity. Absent or severely reduced GlcNAc-1-phosphotransferase activity due to strong alterations of GNPTAB leads to the severe ICD phenotype. Having residual enzyme activity (approximately 10%) due to at least 1 GNPTAB allele being active is associated with the less severe ML III alpha/beta disease.[32][56] Patients with ML III can survive into their adulthood, whereas patients with ML II often die within the first decade of life. ML III is divided into ML IIIA (pseudo-Hurler polydystrophy) and ML IIIC (variant pseudo-Hurler polydystrophy). In ML IIIA, the G1cNAc-1-phosphotransferase activity is reduced. In ML IIIC, G1cNAc-1-phosphate transfer to alpha-methylmannoside is normal, but the transfer to lysosomal substrates is reduced.[57] Measuring G1cNAc-1-phosphotransferase levels can help differentiate ML II from other mucopolysaccharidoses. The lysosomal enzymes, lacking the M6P signal, cannot be sensed by the receptors in the TGN, so further targeting of the endosome/lysosome system cannot be carried out.[72] The progression of protein targeting is due to the cargo-loaded and ligand-bound carrier vesicles. The receptors remain to wait for the signaling ligand to continue the trafficking process. Since the ligand never arrives, the unbound receptors are carried by a group of vesicles, different from the carrier vesicles, to undergo exocytosis.[34] Braulke et al found that I-cell fibroblasts have twice as many MPRs and a higher affinity for M6P and IGF II than normal fibroblasts. The increase in receptors is due to increased mRNA expression of MPRs in I-cells. The receptor count in I-cells remained relatively stable under the influence of substances that increased the surface receptor count.[37] The MPRs are found mainly in the Golgi apparatus with a reduced or absent concentration on the path to and in the endosomes/lysosomes.[34]

The lysosomal enzymes, lacking the M6P signal, cannot be sensed by the receptors in the TGN, so further targeting of the endosome/lysosome system cannot be carried out.[72] The progression of protein targeting is due to the cargo-loaded and ligand-bound carrier vesicles. The receptors remain to wait for the signaling ligand to continue the trafficking process. Since the ligand never arrives, the unbound receptors are carried by a group of vesicles, different from the carrier vesicles, to undergo exocytosis.[34] Braulke et al found that I-cell fibroblasts have twice as many MPRs and a higher affinity for M6P and IGF II than normal fibroblasts. The increase in receptors is due to increased mRNA expression of MPRs in I-cells. The receptor count in I-cells remained relatively stable under the influence of substances that increased the surface receptor count.[37] The MPRs are found mainly in the Golgi apparatus with a reduced or absent concentration on the path to and in the endosomes/lysosomes.[34] In ICD, lysosomal enzymes like glycosidases and sulfatases, among several others, are improperly sorted and targeted, leading to their extracellular accumulation. Lysosomes become dysfunctional and enlarged due to nondegradable macromolecules such as cholesterol, phospholipids, and glycosaminoglycans. This disrupts cellular functioning and homeostasis.[33] Exogenous hydrolase uptake by lysosomes may also be affected due to altered M6P carbohydrate chain or residue recognition markers on the hydrolase molecule.[67][36] The inclusions of ICD have been described extensively from microscopic studies of cultured fibroblasts.[73] Two types of cytoplasmic inclusions have been described: Type 1 and Type 2. Type 1 inclusions are predominantly clear, membrane-bound vesicles of 0.5 to 2.0 micrometers in diameter. Type 2 inclusions contain abundant polymorphic material and concentric globules, membrane-bound and similar in size.[59]

In ICD, lysosomal enzymes like glycosidases and sulfatases, among several others, are improperly sorted and targeted, leading to their extracellular accumulation. Lysosomes become dysfunctional and enlarged due to nondegradable macromolecules such as cholesterol, phospholipids, and glycosaminoglycans. This disrupts cellular functioning and homeostasis.[33] Exogenous hydrolase uptake by lysosomes may also be affected due to altered M6P carbohydrate chain or residue recognition markers on the hydrolase molecule.[67][36] The inclusions of ICD have been described extensively from microscopic studies of cultured fibroblasts.[73] Two types of cytoplasmic inclusions have been described: Type 1 and Type 2. Type 1 inclusions are predominantly clear, membrane-bound vesicles of 0.5 to 2.0 micrometers in diameter. Type 2 inclusions contain abundant polymorphic material and concentric globules, membrane-bound and similar in size.[59] Both types of inclusions are often found within the same cell. In the study by Aula et al, fibroblasts, primitive mesenchymal cells like macrophages and endothelial and perithelial cells contained the inclusions, whereas epithelial and glandular cells did not. A peculiarity was found by identifying numerous type 1 inclusions and some type 2 inclusions in the glomerular podocytes of the kidney, which are differentiated epithelial cells.[59] In the 2019 study by Yokoi et al, 3 patients with ICD only had inclusions in B cells without any inclusions in CD4 T-cells, CD8 T-cells, natural killer cells, monocytes, or neutrophils. The B cell inclusions contained HLA class II molecules. This finding suggests that G1cNAc-1-phosphotransferase has an immune function.[74] Apoptosis

Both types of inclusions are often found within the same cell. In the study by Aula et al, fibroblasts, primitive mesenchymal cells like macrophages and endothelial and perithelial cells contained the inclusions, whereas epithelial and glandular cells did not. A peculiarity was found by identifying numerous type 1 inclusions and some type 2 inclusions in the glomerular podocytes of the kidney, which are differentiated epithelial cells.[59] In the 2019 study by Yokoi et al, 3 patients with ICD only had inclusions in B cells without any inclusions in CD4 T-cells, CD8 T-cells, natural killer cells, monocytes, or neutrophils. The B cell inclusions contained HLA class II molecules. This finding suggests that G1cNAc-1-phosphotransferase has an immune function.[74] Apoptosis Apoptosis is programmed cell death that can occur via 2 different pathways (mitochondrial and death receptor pathways). Lysosomes play a role in the progression of apoptosis, particularly with the release of proteases such as cathepsin D.[75] As a result of the misdirection of various lysosomal enzymes in ICD, apoptosis still occurs but is delayed. This is related to the lower activities of cathepsins D, B, and L in I-cells compared to normal fibroblasts, as found by Terman et al. The same group performed experiments that assessed I-cells' ability to undergo apoptosis compared to normal fibroblasts. Their group found that apoptotic inducers such as MSDH, staurosporine, and naphthazarin had a decreased effect on I-cells. The self-destruction process was reduced when using apoptotic inhibitors leupeptin and pepstatin A on staurosporine-treated normal fibroblasts. No significant change was noted when the same experiment was performed on I-cells.[72]