User:Emilyhagg26/sandbox

The mannose 6-phophate receptors (MPRs) are transmembrane glycoproteins that target enzymes to the lysosomes in vertebrates. There are 2 distinct MPRs: the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor (CD-MPR). Both the CI-MPR and CD-MPR are members of the P-type lectin family which recognise and bind terminal mannose 6-phosphate (Man 6-phosphate) residues.

History
Elizabeth Neufeld was studying patients who had multiple inclusion bodies present in their cells. Due to the large amount of inclusion bodies she named this condition I-cell disease. These inclusion bodies represented lysosomes that were filled with undigestable material. At first Neufeld thought these patients must have a lack of lysosomal enzymes. Further study showed that all of the lysosomal enzymes were being made but they were being secreted rather than being targeted to the lysosome. It was discovered that these enzymes were not phosphorylated. Therefore Neufeld suggested that I-cell disease was caused by a deficiency in the enzymes that add a specific Man 6-phosphate tag onto lysosomal enzymes so they can be targeted to the lysosome.

Studies of I-cell disease led to the discovery of the receptors that bind to this specific tag. Firstly the CI-MPR was discovered and isolated through the use of affinity chromatography. However scientists discovered that some of the lysosomal enzymes still reached the lysosome in the absence of the CI-MPR. This led to the identification of another Man 6-phosphate binding receptor, the CD-MPR, which binds its ligand in the presence of a divalent cation such as Mn2+.

The genes for each receptor have been cloned and characterised. It is thought that they have evolved from the same ancestral gene as there is conservation in some of their intron/ exon borders and there is a homology in their binding domains.

Mechanism of targeting
Lysosomal enzymes are synthesised in the rough endoplasmic reticulum along with a range of other secretory proteins. A specific recognition tag has evolved to prevent these harmful lysosomal enzymes from being secreted and to ensure they are targeted to the lysosome. This tag is a Man 6-phosphate residue.

Once the lysosomal enzyme has been translocated into the rough endoplasmic reticulum an oligosaccharide comprised of Glc3Man9GlcNAc2 is transferred en bloc to the protein. The oligosaccharide present on lysosomal enzymes is processed in the same manner as other secretory proteins whilst it is translocated from the endoplasmic reticulum to the cis-Golgi.



In the cis-Golgi a GlcNAc phosphotransferase (EC 2.7.8.17) adds a GlcNAc-1-phosphate residue onto the 6-hydroxyl group of a specific mannose residue within the oligosaccharide. This forms a phosphodiester: Man-phosphate-GlcNAc. Once a phosphodiester has been formed the lysosomal enzyme will be translocated through the Golgi apparatus to the trans-Golgi. In the trans-Golgi a phosphodiesterase (EC 3.1.4.45) will remove the GlcNAc residue to leave behind Man 6-phosphate. Once Man 6-phosphate has been uncovered the lysosomal enzymes are now able to bind to the CI-MPR and the CD-MPR. The MPR-lysosomal enzyme complex is translocated to a pre-lysosomal compartment, known as endosomes, in a clathrin-coated vesicle. This targeting away from the secretory pathway is achieved by the presence of a specific sorting signal, an acidic cluster/dileucine motif, in the cytoplasmic tails of the MPRs. Both MPRs bind their ligands most effectively at pH 6 – 7; thus enabling the receptors to bind to the lysosomal enzymes in the trans-Golgi and release them in the acidified environment of the endosome. Once the enzyme has dissociated from the MPR it is translocated to the lysosome. Upon arrival in the lysosome the phosphate tag is removed from the enzyme.

MPRs are not found in the lysosomes; they cycle mainly between the trans-Golgi network and endosomes. The CI-MPR is also present on the cell surface. Around 10-20% of the CI-MPR can be found at the cell membrane. Its function here is to capture any Man 6-phosphate tagged enzymes that have accidentally entered the secretory pathway. Once it binds to a lysosomal enzyme the receptor become internalised rapidly. Internalisation is mediated by a sorting signal in its cytoplasmic tail – a YSKV motif. This ensures that all harmful lysosomal enzymes will be targeted to the lysosome.

Knockout mice studies
CI-MPR

Mice lacking the CI-MPR die at day 15 of gestation due to cardiac hyperplasia. The mice suffer from abnormal growth because they are unable to regulate the levels of free IGF-II (insulin-like growth factor type II). Death of the mice can be prevented if the IGF-II allele is also knocked out. Further analysis of the embryos also shows that they display defects in the targeting of lysosomal enzymes as they have an increased level of phosphorylated lysosomal enzymes in their amniotic fluid. Approximately 70% of lysosomal enzymes are secreted in the absence of the CI-MPR – this suggests that the CD-MPR is unable to compensate for its loss.

CD-MPR

When the CD-MPR is knocked out in mice we observe mice that appear healthy apart from the fact that they have defects in the targeting of multiple lysosomal enzymes. These mice display elevated levels of phosphorylated lysosomal enzymes in their blood and they accumulate undigested material in their lysosomes.

From these knockout mice we can deduce that both receptors are needed for the efficient targeting of lysosomal enzymes. If we compare the lysosomal enzymes that are secreted by the two different knockout cell lines we see different sets of enzymes. This suggests that each MPR interacts preferentially with a subset of lysosomal enzymes.

Structure
The CI-MPR and CD-MPR are structurally distinct receptors however they share an overall general structure as they are both type I integral membrane proteins. Both receptors have a large N-terminal extracytoplasmic domain, one transmembrane domain and a short C-terminal cytoplasmic tail. These cytoplasmic tails contain multiple sorting signals ; some of which can be either phosphorylated or palmitoylated.



CI-MPR: The CI-MPR is ~300kDa. The N-terminal extracytoplasmic domain contains 15 contiguous P-type carbohydrate recognition domains. They are referred to as MRH (mannose 6-phosphate receptor homology) domains. The domains are homologous because they have: The structure of 7 out of the 15 domains has been determined, using X-ray crystallography, and they seem to share a similar fold. The CI-MPR exists mainly as a dimer in the membrane. Domains 3, 5 and 9 have been found to bind to Man 6-phosphate. Domains 3 and 9 can bind to Man 6-phosphate with high affinity. Domain 5 only binds Man-6-phosphate with a weak affinity. However domain 5 has also been shown to bind to the phosphodiester, Man-phosphate-GlcNAc. This is a safety mechanism for the cell – it means it is able to bind to lysosomal enzymes that have escaped the action of the enzyme that removes the GlcNAc residue. Combining these 3 domains allows the CI-MPR to bind to a wide range of phosphorylated glycan structures. Domain 11 binds to IGF-II.
 * A similar size - each one has around 150 amino acid residues
 * Conserved amino acid residues – between 14-38% sequence identity
 * Conserved positioning of 6 specific Cysteine residues that are involved in forming disulphide bonds

CD-MPR: The CD-MPR is much smaller than the CI-MPR – it is only ~46kDa. Its N-terminal extracytoplasmic domain contains only 1 P-type carbohydrate recognition domain. The CD-MPR exists mainly as a dimer in the membrane. However monomeric and tetrameric forms are also thought to exist as well. The equilibrium between these different oligomers is affected by pH, temperature and presence of Man 6-phosphate residues. Each monomer forms a 9 stranded β-barrel which can bind to a single Man 6-phosphate residue.

Mannose 6-phosphate Binding
The CI-MPR and CD-MPR bind Man 6-phosphate in a similar fashion. Both form a set of hydrogen bonds between key residues and characteristic hydroxyl groups on the mannose residue. Hydrogen bonds to hydroxyl groups at positions 2, 3 and 4 make the site specific for mannose alone.

Both MPRs share 4 residues that are essential for ligand binding. Mutation of any of these residues results in the loss of Man 6-phosphate binding. These residues are glutamine, arginine, glutamic acid and tyrosine and are responsible for forming the hydrogen bonds that contact specific hydroxyl groups in the mannose residue.

A wide range of N-glycan structures can be present on lysosomal enzymes. These glycans can vary in: The CI-MPR and CD-MPR are able to bind to this wide range of N-glycan structures by having a different binding site architecture. The MPRs also bind to the phosphate group in a slightly different manner. Domain 3 of the CI-MPR uses Ser-386 and an ordered water molecule to bind to the phosphate moiety. On the other hand the CD-MPR uses residues Asp-103, Asn-104 and His-105 to form favourable hydrogen bonds to the phosphate group. The CD-MPR also contains a divalent cation Mn2+ which forms favourable hydrogen bonds with the phosphate moiety.
 * Type – hybrid or high mannose structures
 * Size
 * Presence of the phosphomonoester (Man 6-phosphate) or phosphodiester (Man-phosphate-GlcNAc)
 * Number of Man 6-phosphate tags
 * Location of the Man 6-phosphate tag

CI-MPR and Cancer
It is well-established that the CI-MPR binds Man 6-phosphate but there is a growing body of evidence that suggests the CI-MPR also binds to unglycosylated IGF-II. It is thought that when the CI-MPR is present on the cell surface, domain 11 will bind to any IGF-II free in the extracellular matrix. The receptor is then rapidly internalised, along with IGF-II, through a YSKV motif present in the CI-MPR’s cytoplasmic tail. IGF-II will then be targeted to the lysosome where it will be degraded. This regulates the level of free IGF-II in the body.

This function of the CI-MPR was determined through the use of knockout mice. It was observed that CI-MPR deficient mice had an increased level of free IGF-II and enlarged organs (around a 30% increase in size ). These mice die at day 15 of gestation due to cardiac hyperplasia. Death of the mice could be prevented when the IGF-II allele was also knocked out. When the CI-MPR and the IGF-II allele are knocked out we see a normal mouse as there is no longer a growth factor present that needs to be regulated.

Due to CI-MPR’s ability to modulate the levels of IGF-II it has been suggested it may play a role as a tumour suppressor. Studies of multiple human cancers have shown that a loss of the CI-MPR function is associated with a progression in tumourigenesis. Loss of heterozygosity (LOH) at the CI-MPR locus has been displayed in multiple cancer types including liver and breast. However this is a relatively new concept and many more studies will have to investigate the relationship between the CI-MPR and cancer.