Biochemistry and Genetics
The gene mutated in MLIV has been named MCOLN1. It contains 14 exons and spans 12,300 base pairs on chromosome 19p13.3 (Bargal et al, 2000; Bassi et al, 2000; Sun et al, 2000). The product of this gene is called mucolipin-1, a 580-amino-acid protein with 6 transmembrane domains that shares homology to the transient receptor potential channels and other nonselective cation channels (Fig. 138.1-5) (Clapham, 2003). TRP proteins function as membrane sensors in many types of cells, and they are responsible for several crucial signal transduction mechanisms (Chen et al, 2001; Gonzalez-Perrett et al, 2001; Hanaoka et al, 2000; LaPlante et al, 2002; Raychowdhury et al, 2004; Sun et al, 2000; Vassilev et al, 2001). MCOLN1 is very similar to two other human genes, MCOLN2 and MCOLN3, and together they make up the TRPML subfamily of the TRP superfamily (Montell, 2001). Interestingly, MCOLN2 and MCOLN3 map adjacent to each other on chromosome 1p21, and the gene family is the result of gene duplication (Di Palma et al, 2002; Sun et al, 2000). All three members of the gene family are present in zebra fish (Danio rerio), and the proteins show a high degree of homology to the human mucolipins (mucolipin-1, 61% identity and 75% similarity; mucolipin-2, 59% identity and 75% similarity; mucolipin-3, 62% identity and 81% similarity). Invertebrates have only one mucolipin gene, which has been identified in Caenorhabditis elegans (CUP-5) and in several insects, including Drosophila. Mucolipin-3 was found to be mutated in the Varitint-Waddler mouse, which serves as a model for deafness and pigmentation defects (Di Palma et al, 2002). The human mucolipins are closely related to polycystin-2, which is mutated in autosomal dominant polycystic kidney disease (Sun et al, 2000). Polycystin-2 and mucolipin-1 are known to function as nonselective cation channels and are believed to be calcium channels (Chen et al, 2001; Gonzalez-Perrett et al, 2001; Hanaoka et al, 2000; LaPlante et al, 2002; Raychowdhury et al, 2004; Vassilev et al, 2001).
Mucolipin-1 predicted structure and MLIV-causing mutations in the protein. Mucolipin-1 has 6 predicted transmembrane domains (barrels) with the N and the C terminals facing inward toward the cytosolic side. It has a big 200-amino-acid-long loop between the first and second transmembrane domains; the loop is common to mucolipins and to polycystin-2, which otherwise has a low similarity to mucolipin in its amino acid sequence but a very high structural similarity. The regions that are similar between mucolipin-1, -2, and -3 are highlighted in pale blue, and the section that is similar to other TRP channels or the TRP domain PS50272 is highlighted in pale green. This section also contains the predicted channel pore (red arrow). Yellow arrows point to MLIV-causing mutations that are predicted to change the protein product. Many mutations surround the channel pore or the transmembrane domains in the TRP region. None appears in the terminal cytosolic tails.
It is unclear why a deficiency or malfunction of mucolipin-1 causes MLIV. Electrophysiologic studies with mucolipin-1 constructs containing known MLIV mutations suggest that they may reduce channel function or affect the regulation of the channel by pH (Raychowdhury et al, 2004). Studies with mutated mucolipin in C. elegans coelomocytes suggest that mucolipin-1 is involved in the regulation of lysosomal function (Fares, Greenwald, 2001; Treusch et al, in press). Support for this idea can be found in the cytologic studies describing vacuolation of many cell types in MLIV, as some of the vacuoles have been shown to be of late-endosomal/lysosomal nature (Figs. Fig. 138.1-4 and Fig. 138.1-6) (Goldin et al, 1995; Schiffmann et al, 1998; Smith et al, 2002). It is possible that mucolipin-1 is required for calcium currents involved in endocytic membrane trafficking (Luzio et al, 2003), although to date this has not been tested. Perhaps the vacuolation seen in MLIV cells is caused by the destabilization of a membrane protein complex that might result from the absence or malfunction of mucolipin-1. We speculate that in addition to its action as a cation channel, mucolipin-1 may also hold a functional membrane unit together. Given its absence in MLIV, perhaps the cell is trying to compensate by increasing the rate of membrane recycling and internalization into lysosomes, thereby causing a traffic jam at the endosomal-lysosomal junction. Conceivably, a combination of both channel activity and the membrane stabilization functions of mucolipin-1 is required in most normals. However, many cell types would survive virtually unaffected without the protein, and some will do well with a mutated protein that is only partially functional. Partial functionality might explain the difference in disease phenotype between patients with no expression of mucolipin-1 and those with production of a mutant protein. Typically, patients with production of mutant mucolipin-1 display a milder phenotype than do those with complete absence (Altarescu et al, 2002). This is modeled nicely in C. elegans , in which the point mutation ar465 G401E, which is identical to G368 in human mucolipin-1, is viable while the complete absence of the protein product in zu223 is embryonic lethal (Fares, Greenwald, 2001; Hersh et al, 2002). Based on the patients identified to date, we hypothesize that a partially functional mucolipin-1, for example ΔF408, may cause only degenerative defects such as loss of vision and cerebellar motor control (Altarescu et al, 2002). One function that clearly depends on the fully active mucolipin-1 is acid secretion, since it is absent in even minimally affected patients (Schiffmann et al, 1998). Lack of acid secretion could be attributed to the involvement of mucolipin-1 in trafficking of membranes. MLIV parietal cells seem to lack tubulovesicles, endosomal entities that harbor the acid secretion complex in quiescent cells (Schiffmann et al, 1998). Lack of mucolipin-1 could prevent formation of these structures or cause their fusion with lysosomes, which are prominently enlarged in MLIV parietal cells (Fig. 138.1-4 and Fig. 138.1-6) (Lubensky et al, 1999; Schiffmann et al, 1998). However, it is also possible that mucolipin directly regulates the triggering of acid secretion, and in its absence, the cell tries to compensate by excessive recycling of apical membranes--hence the formation of vacuoles. There are four mouse models of achlorhydria, with only those that are knockout for the α or β subunits of the H+/K+-ATPase showing vacuolation of the parietal cells (Gawenis et al, 2004; Samuelson, Hinkle, 2003). Autofluorescence in cultured fibroblasts is also present in all MLIV patient cell lines examined to date (Fig. 138.1-7) (Goldin et al, 1999). It is probably associated with the vacuolation of lysosomes and is more prominent in severely affected patients (Ehud Goldin, unpublished observation).
An electron micrograph of a parietal cell from stomach of a patient with MLIV. The parietal cell lysosomes (Ly) are large and contain structurally heterogeneous material including vesicles (V) and masses of lamellar structure (L), and mitochondria (M) typical for parietal cells (original magnification ×6500). (From Schiffmann et al, 1998.)
Elimination of autofluorescence in fusion products of normal and MLIV fibroblasts. Normal (stained green) and MLIV (stained red) fibroblasts were fused as previously described (Schiffmann et al, 1998). Four days after seeding on slides, cells were fixed and photographed. A. A phase micrograph showing a normal cell (nucleus marked N), a normal-MLIV fusion product (nuclei marked N, M), and an MLIV cell (nucleus marked M). The large arrow points to the phase-dense material. B. Fluorescence in the UV channel showing autofluorescence in MLIV cell and not in the fusion product. The autofluorescence colocalizes with the phase-dense material. C. and D. demonstrated that the fused cell on the right of the panels is a product of a fusion of a normal cell (the N nucleus is green in C.) and an MLIV cell that has nucleus stained in red (M in D.). In C., fluorescence in the green wavelength in mutant cell reflects only the autofluorescent material that is seen in B.
Increased membrane recycling would explain the increased rate of uptake of fluid phase markers in mucolipin-defective C. elegans coelomocytes; it would also explain the increased rate of uptake of certain compounds in human MLIV fibroblasts as well as the increased rate of membrane synthesis (Bach, Desnick, 1988; Chen et al, 1998; Kopitz et al, 1994; Pagano, Chen, 1998). The pathologic changes caused by MLIV also affect the function of other proteins that probably share with mucolipin-1 a currently unknown pathway. An example is protein kinase C, which was found to have reduced phosphorylation in MLIV fibroblast membrane fractions (Boneh, Bach, 1993; Turgeman, Boneh, 1996). Reduction in growth in MLIV patients is likely associated with a reduction of expression of insulin-like growth factor binding protein 3 (IGFBP3) and secondarily in insulin-like growth factor (IGF) (Ehud Goldin, unpublished observations). It is possible that the protein complex held at the membrane by mucolipin-1 involves other proteins in the IGF pathway or that the changes in that pathway are compensatory to the loss of function of mucolipin-1. Mucolipin-1 is expressed in most human tissues. In C. elegans it is also ubiquitous. In Drosophila, however, the expression of mucolipin is confined to the salivary glands, the midgut, and the malpighian tubules at specific embryonic stages (Fig. 138.1-8) (Ward Odenwald, unpublished observations). This is in accord with its predicted role in the human stomach. Over expression of tagged mucolipin-1 in cultured cells showed that it was found mainly in fractions that contain late endosomes and lysosomes (Raychowdhury MK, et al. Molecular pathophysiology of mucolipidosis type IV: pH dysregulation of the mucolipin-1 cation channel. Hum Mol Genet. 2004; 13:617-27). However, this localization might be an artifact due to overexpression. Precise localization will require the production of mucolipin-1-specific antibodies, which have not yet been described. Several putative lysosomal targeting domains on mucolipin-1 are currently being investigated to test their relevance in targeting the protein. Perhaps the majority of the protein will be found integrated in intracellular membranes, while the functional complex may be targeted to the plasma membrane as was demonstrated in the case of the polycystin-1 and polycystin-2 complexes (Hanaoka et al, 2000). To date, there is no evidence for a specific complex formed by mucolipin-1 and other membrane proteins, although many channel proteins of the six-transmembrane domain composition do form hetero- or homo-oligomers of four subunits (Clapham, 2003). The fact that the phenotype of MLIV has not been found with a defect in a different gene supports the idea that if it does complex with other specific proteins, mutations in those will result in a different disease phenotype (Goldin et al, 1999).
In situ hybridization staining of mucolipin in Drosophila embryos. Embryos at different ages were stained with a Drosophila mucolipin gene probe. Images represent embryos of increasing ages. Left to right, an egg with diffuse maternal mRNA; blastocyte with reduced maternal expression; staining of cells surrounding the amnioserosa at a later stage; appearance of specific staining in the gut (G), malpighian tubules (M), and salivary glands (S); and increased staining at stage 13 of the embryo.
The mutations identified so far in MCOLN1 in MLIV patients are described in Table 138.1-1. A variety of mutations were found to cause MLIV, including splice mutations, small and large deletions and insertions, and point mutations that cause either stop codons or amino acid changes in mucolipin-1. The two most prevalent mutations are responsible for the majority of MLIV patients in the Ashkenazi-Jewish population. One is the splice mutation g.5534A→G (Table 138.1-1) that prevents splicing of mucolipin-1 mRNA at exon 4, resulting in a mix of unstable aberrant mRNA species. The second is a deletion mutation that eliminates 6434 bp of DNA from chromosome 19, including the first 7 exons of MCOLN1. While the combined carrier frequency of these mutations ranges from 1/62 to 1/127 in individuals of Ashkenazi Jewish descent (Bargal et al, 2001; Edelmann et al, 2002; Wang et al, 2001), the splice mutation is at least three times more common than the deletion mutation (Edelmann et al, 2002). The deletion mutation is particularly rare in the Israeli population (1/2000) in comparison to its frequency in the New York metropolitan area (1/406) (Bargal et al, 2001; Edelmann et al, 2002). About one-third of the MLIV patients of Ashkenazi Jewish ancestry in the United States are compound heterozygotes for both mutations (Ehud Goldin, unpublished; Wang et al, 2001). Thus far, only one patient homozygous for the deletion mutation has been identified (Bargal et al, 2000; Bassi et al, 2000; Sun et al, 2000). A Polish patient with a non-Jewish haplotype was found to be heterozygous for this mutation (Sun et al, 2000). A splice mutation that causes a deletion of 15 bp near the 3′ end of mucolipin (c.1406A→G) was discovered in a Canadian family from Newfoundland (Table 138.1-1). This mutation causes a moderate form of MLIV, as the patients are able to walk independently and have better communicative skills (Altarescu et al, 2002). A second Canadian family was found to carry a 93-bp insertion mutation in exon 2 that leads to altered splicing and null expression. The inserted segment was identical in sequence to part of the gene encoding mitochondrial NADH dehydrogenase subunit 5 (Goldin et al, 2004). An 11-nucleotide insertion mutation causing a frameshift in exon 10 was discovered in 2 non-Jewish patients (Altarescu et al, 2002; Bargal et al, 2001). Missense mutations were found in the loop between the first and second transmembrane domain: 1 in the lipase domain and 1 eliminating the 4 cysteines in the loop, possibly reducing the stability of mucolipin (Table 138.1-1 and Fig. 138.1-5). These patients had a milder phenotype, had an independent ataxic gait, and could feed themselves with their hands. In several patients from the southeast United States we found a D362Y amino acid change in the third transmembrane domain. This mutation was associated with a slower progression of retinal disease and a relatively mild neurologic phenotype, although membrane preparations containing mucolipin-1 with this mutation had no channel activity (Raychowdhury et al, 2004). Several mutations were discovered in the fourth transmembrane domain, including F408del, which causes the mildest MLIV phenotype known (Fig. 138.1-5) (Altarescu et al, 2002). The protein construct containing this mutation still functions as a channel in liposome preparations and only displays a deficiency in regulation (Raychowdhury et al, 2004). Several other mutations were discovered in the area of the presumed channel pore between the fifth and sixth transmembrane domain. Most of those were associated with a more severe MLIV phenotype (Table 138.1-1) (Altarescu et al, 2002).
Table 138.1-1: MCOLN1 Mutations |Favorite Table|Download (.pdf) Table 138.1-1: MCOLN1 Mutations
| Mutation Number || Nucleotide Changea || Mutation Type || AA Change || Mutation Ancestry || Patient Genotypeb || Reference |
|1 ||g.5534A→G ||Splicec ||– ||AJ || ||Bargal et al, 2000; Bassi et al, 2000; Sun et al, 2000 |
|2 ||c.1406A→G ||Splicec ||454-469del ||NJC (CA) ||2,2 ||Sun et al, 2000 |
|3 ||g.511-6944del ||6434-bp deletion ||– ||AJ || ||Bargal et al, 2000 |
|4 ||c.163_197del, c.163_197insTCA ||Frameshiftc ||– ||NJC (G) ||4,4 ||Bargal et al, 2002 |
|5 ||c.1221_1223delCTT ||aa del ||F40Sdel ||AJ ||1,5 ||Bargal et al, 2000; Sun et al, 2000 |
|6 ||c.473_474delCC ||Frameshiftc ||– ||NJC (P) ||3,6 ||Sun et al, 2000 |
|7 ||c.1209-1210insT c.1463_1464insGGCCGCAGCAG ||Frameshiftc ||– ||AJ ||3,7 ||Bargal et al, 2000; Sun et al, 2000 |
|8 ||G ||Frameshiftc ||– ||NJC || ||Altarescu et al, 2002 |
|9 ||c.304C→T ||Nonsense ||R102X ||NJC ||9,15 ||Sun et al, 2000 |
|10 ||c.514C→T ||Nonsense ||R172X ||NJC (CA) ||2,10 ||Sun et al, 2000 |
|11 ||c.964C→T ||Nonsense ||R322X ||NJ (AD) ||1,11 ||Bargal et al, 2001 |
|12 ||c.317T→C ||Missense ||L106P ||NJC ||12,18 ||Altarescu et al, 2002 |
|13 ||c.497G→T ||Missense ||C166F ||AA ||13,? ||Goldin, unpublished |
|14 ||c.694A→C ||Missense ||T232P ||NJ ||3,14 ||Bargal et al, 2001 |
|15 ||c.1084G→T ||Missense ||D362Y ||NJC || ||Sun et al, 2000 |
|16 ||c.1207C→T ||Missense ||R403C ||NJC(CA) ||16,20 ||Goldin, unpublished |
|17 ||c.1336G→T ||Missense ||V446L ||HA ||17,17 ||Sun et al, 2000 |
|18 ||c.1340T→C ||Missense ||L447P ||NJC ||12,18 ||Altarescu et al, 2002 |
|19 ||c.1395C→G ||Missense ||F465L ||NJ ||1,19 ||Bargal et al, 2001 |
|20 ||c.1388G→A ||Missense ||C463Y ||NJ || ||Goldin, unpublished |
|21 ||c.236_237ins93 from NADH dehydrogenase 5 99-192 ||31 aa insertion |
|In-frame segment of NADHc dehydrogenase 5 ||NJC (CA) ||16,20 ||Goldin et al, 2004 |