Lysosomes are intracellular membrane-bound organelles that perform the final degradation of many cellular macromolecules. This is achieved by the action of a number of lysosomal enzymes (originally called “acid hydrolases” because of the low internal pH characteristic of lysosomes). These enzymes are synthesized in the endoplasmic reticulum (ER) on membrane-bound ribosomes and traverse the ER-Golgi pathway along with other newly synthesized proteins. At the terminal Golgi compartment (the trans-Golgi network or TGN), they are segregated from all other glycoproteins and selectively delivered to lysosomes. In most “higher” animal cells, this specialized trafficking is achieved primarily by a specific glycan marker that is recognized by certain receptors. This chapter describes the discovery and characterization of this glycan-mediated biological system, which relies on recognition of glycans containing mannose-6-phosphate (M6P) by “P-type” lectins. This was the first clear-cut example of a biological role for glycans on mammalian glycoproteins and the first demonstrated link between glycoprotein biosynthesis and human disease. The interesting history of its discovery is therefore described in some detail.

HISTORICAL BACKGROUND

I-Cell Disease and the “Common Recognition Marker” of Lysosomal Enzymes

Early studies of human genetic “storage disorders” by Elizabeth Neufeld and colleagues indicated a failure of intracellular degradation of cellular components that therefore accumulated in lysosomes (see Chapter 41). Soluble “corrective factors” from normal cells could reverse these defects when added to the culture media. These factors were found to be lysosomal enzymes, and they were deficient in patients with different diseases. They were secreted in small quantities by normal cells in culture (or by cells from patients with a different “complementary” defect) (Figure 30.1). The enzymes existed in two forms: a “high-uptake” form, recognized by saturable, high-affinity receptors that could correct deficient cells, and an inactive “low-uptake” form that could not correct the defect. Meanwhile, fibroblasts from patients with a rare human genetic disease exhibiting prominent inclusion bodies in cultured cells (therefore termed “I-cell” disease) were found to be deficient in not one, but almost all, lysosomal enzymes. In I-cells, all of the lysosomal enzymes are actually made, but they are almost completely secreted into the medium instead of being retained in the lysosomes. Although I-cells can incorporate the high-uptake enzymes secreted by normal cells, enzyme molecules secreted by I-cells are not taken up by other cells (Figure 30.1). Neufeld therefore proposed that I-cell disease results from a failure to add a “common recognition marker” to all lysosomal enzymes. This marker was assumed to be responsible for the retention of lysosomal enzymes in normal cells and also for internal trafficking of the enzymes to lysosomes. Because the high-uptake property was destroyed by periodate treatment, it was predicted that the recognition marker was a glycan.

FIGURE 30.1

Historical background regarding “cross-correction” of lysosomal enzyme deficiencies in cultured cells. Small amounts of “high-uptake” lysosomal enzymes secreted by normal fibroblasts (thin arrows) were found to be taken up (more...)

Enzymatic Mechanism for Generation of the M6P Recognition Marker

Correction

In the section “Enzymatic Mechanism for Generation of the M6P Recognition Marker,” “Uridine-P+GlcNAcα1-6-Manα1-(N-glycan)-Lysosomal enzyme” was changed to “Uridine-P+GlcNAcα1-P-6-Manα1-(N-glycan)-Lysosomal enzyme” in the second reaction of the biochemical pathway graphic.

Oligomannosyl N-glycans released from lysosomal enzymes were found to carry one or two phosphate residues at the C-6 of various mannose residues. Comparison of glycans with phosphodiesters and phosphomonoesters predicted that the metabolic precursor was a phosphodiester and that phosphorylation was mediated not by an ATP-dependent kinase, but by a UDP-GlcNAc-dependent GlcNAc-1-phosphotransferase. This was proven using a double-labeled donor substrate and Golgi extracts:

Reactants: Uridine-P-³²P-[6-³H]GlcNAc + Manα1-(N-glycan)-lysosomal enzyme

Products: Uridine-P + [6-³H]GlcNAcα1-³²P-6-Manα1-(N-glycan)-lysosomal enzyme

Another Golgi enzyme was shown to remove the outer N-acetylglucosamine residue and “uncover” the phosphomonoester, generating the M6P moiety. Pulse-chase studies confirmed the order of events, indicating that more than one glycan on a given lysosomal enzyme could be phosphorylated and that removal of some mannose residues on the N-glycan by processing Golgi mannosidases was also required (Figure 30.2). In most cell types, the phosphate is eventually lost from the M6P, presumably after exposure to acid phosphatase in lysosomes. Thus, the overall biochemical pathway is as follows:

FIGURE 30.2

Pathways for biosynthesis of N-glycans bearing the mannose-6-phosphate (M6P) recognition marker. Following early N-glycan processing (see Chapter 8 for details), a single GlcNAc phosphodiester is added to the N-glycans of lysosomal enzymes on one of three (more...)

$$\begin{array}{c}\text{Uridine}-\text{P}-\text{PGlcNAc}+\text{Man}\alpha 1-(\text{N}-\text{glycan})-\text{Lysosomal}\hspace{0.17em}\text{enzyme}\\ \downarrow \hspace{0.17em}\hspace{0.17em}\hspace{0.17em}"Phosphotransferase"\\ \text{Uridine}-\text{P}+\text{GlcNAc}\alpha 1-\text{P}-6-\text{Man}\alpha 1-(\text{N}-\text{glycan})-\text{Lysosomal}\hspace{0.17em}\text{enzyme}\\ \downarrow \hspace{0.17em}\hspace{0.17em}\hspace{0.17em}"Uncovering\hspace{0.17em}Enzyme"\\ \text{GlcNAc}+\text{P}-6-\text{Man}\alpha 1-(\text{N}-\text{glycan})-\text{Lysosomal}\hspace{0.17em}\text{enzyme}\\ \downarrow \hspace{0.17em}\hspace{0.17em}\hspace{0.17em}Lysosomal\hspace{0.17em}Phosphatase\\ \text{P}+\text{Man}\alpha 1-(\text{N}-\text{glycan})-\text{Lysosomal}\hspace{0.17em}\text{enzyme}\end{array}$$

However, during passage through the Golgi, the phosphate residues partially block the action of processing mannosidases, maintaining the N-glycans in an oligomannosyl form (Figure 30.2). Because M6P is lacking on lysosomal enzymes in I-cell disease (see below), these glycans are likely to be processed in the Golgi, thus explaining why the secreted enzymes in these patients carry more sialylated, complex, N-linked glycans.

The genes encoding both enzymes involved in these reactions have been cloned and characterized. The UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-P-T) is a 540-kD complex composed of two disulfide-linked 166-kD α subunits, two disulfide-linked 51-kD β subunits, and two identical noncovalently associated 56-kD γ subunits. The α and β subunits are encoded by a single gene whose product undergoes proteolysis to give rise to the two subunits, and the γ subunit is encoded by a separate gene. The α subunit was identified as the catalytic subunit by photoaffinity labeling. The second enzyme (α-N-acetylglucosaminyl-1-phosphodiester glycosidase) is a 272-kD complex of four identical 68-kD subunits, arranged as two disulfide-linked homodimers. Unlike other Golgi enzymes, this is a type-I membrane-spanning glycoprotein with its amino terminus in the lumen of the Golgi.

Variants of I-Cell Disease and Pseudo-Hurler Polydystrophy

The simplest in vitro substrate for GlcNAc-P-T is α-methyl mannoside. However, it is a poorer substrate than an oligomannosyl N-glycan, which, in turn, is much poorer than a native lysosomal enzyme. Typical glycoproteins bearing oligomannosyl N-glycans that are not lysosomal enzymes are also poor substrates. Thus, the GlcNAc-P-T enzyme must specifically recognize oligomannosyl N-glycans on lysosomal enzymes in preference to those on other glycoproteins, via a second protein–protein recognition site (Figure 30.3). In most cases of ML-II and -III, assays with α-methyl mannoside and lysosomal acceptors both give concomitant reductions in activity. However, rare cases of ML-III show normal activity with the α-methyl mannoside acceptor, but markedly decreased activity with lysosomal enzyme acceptors. The GlcNAc-P-T enzyme in these patients is present in normal catalytic amounts, but because of a mutation in the γ subunit, it is impaired in the recognition of lysosomal enzymes as appropriate acceptors for phosphorylation. This finding provided a genetic basis for specific recognition of lysosomal enzymes by GlcNAc-P-T (see below).

FIGURE 30.3

Selective recognition of lysosomal enzymes by GlcNAc-P-T. GlcNAc-P-T is a multisubunit enzyme (α₂ β ₂ γ ₂). In normal cells, it has three independent binding sites, one for the N-glycan-M6P substrate, one for the UDP-GlcNAc donor, (more...)

COMMON FEATURES OF P-TYPE LECTINS (M6P RECEPTORS)

The first candidate (~275-kD) receptor for the M6P recognition marker was isolated by affinity chromatography and was found to bind M6P in the absence of cations. Certain cells deficient in this receptor still showed M6P-inhibitable binding of lysosomal enzymes, leading to the discovery of a second M6P receptor (MPR) of approximately 45 kD, which required divalent cations for optimal binding. The larger cation-independent M6P receptor (CI-MPR) binds with highest affinity in a 1:1 stoichiometry to glycans carrying two M6P residues (Figure 30.2, structure C) and poorly to molecules bearing GlcNAc-P-Man phosphodiesters (Figure 30.2, structures A and B). Binding to molecules carrying one M6P (Figure 30.2, structure D) is intermediate in affinity. The smaller, cation-dependent MPR (CD-MPR) has only one binding site for a single M6P. In vitro removal of “blocking” GlcNAc residues from molecules carrying two M6P-GlcNAc residues improved binding to both receptors. Treatment with an α-mannosidase enhanced binding, confirming that removal of outer mannose residues by the processing Golgi mannosidases is also a requirement for recognition. These findings with isolated N-glycans were confirmed and extended by studying their direct uptake into cells.

Genes encoding both MPRs have been cloned and extensively characterized. Both are type I membrane glycoproteins with large extracytoplasmic domains, single transmembrane regions, and relatively small carboxy-terminal cytoplasmic domains. The CI-MPR has 15 unique, contiguous repetitive units of approximately 145 amino acids with partial identity to one another. The CD-MPR has a single extracellular domain, showing homology to the repeating domains of the CI-MPR. Together with conservation of certain intron–exon boundaries, this homology suggests that the two genes evolved from a common ancestor. On the basis of their sequence relationships and unique binding properties to M6P, the two MPRs have been formally classified as P-type lectins. Structural homologs of the MPRs are present in yeast and Drosophila, but these lack M6P-binding ability.

The CD-MPR exists mainly as a dimer, with each monomer binding one M6P residue. However, monomeric and tetrameric forms of the CD-MPR exist, and the equilibrium between forms is affected by temperature, pH, and the presence of ligands. The CI-MPR also seems to be a dimer in the membrane, although it readily dissociates upon solubilization. Somewhat surprisingly, this much larger molecule binds only two residues of M6P, using just 2 of its 15 repeating units (a third repeat binds M6P very weakly). Mutagenesis studies have identified specific residues of these receptors involved in M6P binding, and the crystal structure of the single extracytoplasmic domain of the CD-MPR has been obtained in a complex with M6P. This domain crystallized as a dimer, with each monomer folded into a nine-stranded flattened β-barrel that has a striking resemblance to the protein folds in avidin (Figure 30.4). The distance between the two ligand-binding sites of the dimer provides a good explanation for the differences in binding affinity shown by the CD-MPR toward various lysosomal enzymes. The crystal structure of the amino-terminal 432 residues of the CI-MPR, encompassing domains 1–3 (domain 3 is one of the M6P-binding domains), has also been solved. Each domain exhibits a topology similar to that of the CD-MPR, and the three domains assemble into a compact structure that provides insight into the arrangement of the entire extracellular region of the CI-MPR. The proposed model does not position the two M6P-binding domains (3 and 9) sufficiently close to bind a single, diphosphorylated N-glycan. This suggests that the high-affinity binding of this N-glycan is due to the spanning of binding sites located on different CI-MPR dimers. An interesting possibility is that the receptor is dynamic, with the spacing between the two M6P-binding sites being flexible, to enhance interactions with lysosomal enzymes containing phosphorylated glycans at various positions on their protein backbones.

FIGURE 30.4

Ribbon diagram of the bovine cation-dependent M6P receptor (CD-MPR). Shown are the two monomers (magenta and cyan ribbons) of the dimer as well as the ligand M6P (gold ball-and-stick model). (Modified, with permission, from Roberts et al. 1998. Cell (more...)

GENERATION OF THE “M6P RECOGNITION MARKER” FOR THE MPRS

The M6P recognition marker actually encompasses a family of M6P-bearing N-glycans with varying degrees of affinity for the MPRs, based on the position of phosphate groups and the structure of the underlying N-glycan (Figure 30.2). The number and distribution of such N-glycans on different lysosomal enzymes could further affect binding to the two receptors. Thus, whereas both MPRs have a preference for enzymes containing glycans with two M6P residues, it appears that two appropriately spaced N-glycans with a single M6P each can provide a high-affinity ligand. A cohort of newly synthesized lysosomal enzymes therefore presents a spectrum of affinities for the MPRs. Taken together with factors such as the number, compartmental localization and availability of MPRs, differences in properties of the two receptors, and the concentration of cations, there is clearly much flexibility in this trafficking mechanism. Indeed, different cell types target different M6P-containing proteins to their lysosomes at different rates, with varying proportions being secreted.

NATURAL AND INDUCED GENETIC DEFECTS IN THE MPRS

Targeted disruption of the CD-MPR gene in mice is associated with normal or only slightly elevated levels of lysosomal enzymes in the circulation and an otherwise normal phenotype. However, thymocytes or primary cultured fibroblasts from such mice show an increase in the amount of phosphorylated lysosomal enzymes secreted into the medium. Thus, there must be mechanisms that compensate for the deficiency in vivo. Intravenous injection of inhibitors of other glycan-specific receptors capable of mediating endocytosis (e.g., the mannose receptor of macrophages and the asialoglycoprotein receptor of hepatocytes; see Chapter 31) give rise to a marked increase in lysosomal enzymes in the serum of the deficient mice. Thus, such receptors are likely part of the compensatory mechanisms in vivo.

Other studies have shown that mouse CI-MPR is part of the naturally occurring Tme locus, a maternally imprinted region of chromosome 17 (i.e., expressed only from the maternal chromosome). Mice inheriting a deletion of the Tme locus from their mother die at day 15 of gestation. Genetic disruption of the CI-MPR gene showed that the lethality is due to lack of this receptor. Maternal inheritance of a null allele or homozygosity for the inactive allele is generally lethal at birth, and mutants are about 30% larger in size. The phenotype is probably due to an excess of insulin-like growth factor II (IGF-II), which is another ligand for the CI-MPR (see below), because the introduction of an IGF-II null allele rescued the mutant mice. CI-MPR mutant mice also have organ and skeletal abnormalities.

Cell lines lacking either or both MPRs were obtained by mating CD-MPR–deficient mice with mice heterozygous for a CI-MPR–deleted allele. Like fibroblasts that lack only CD-MPR, fibroblasts that lack only CI-MPR have a partial impairment in sorting. Fibroblasts from embryos that lack both receptors show a massive missorting of multiple lysosomal enzymes and accumulated undigested material in their endocytotic/lysosomal compartments. Thus, both receptors are required for efficient intracellular targeting of lysosomal enzymes. Comparison of lysosomal enzymes secreted by the different cell types indicates that the two receptors may interact preferentially with different subgroups of enzymes. Thus, the structural heterogeneity of the M6P recognition marker within a single lysosomal enzyme and between different enzymes is one explanation for the evolution of two MPRs with complementary binding properties: that is, to provide an efficient but varied targeting of lysosomal proteins in different cell types or tissues. In the final analysis, what initially appeared to be a precise “digital” “lock-and-key” mechanism turns out to be a far more complex and flexible “analog” system.

SUBCELLULAR TRAFFICKING OF THE MPRS

At steady state, MPRs are most concentrated in the TGN and late endosomes, but they cycle constitutively between these organelles, early (sorting) endosomes, recycling endosomes, and the plasma membrane (Figure 30.5). MPRs avoid delivery to lysosomes, where they would be degraded. This trafficking is directed by a number of short amino acid sorting signals in the cytoplasmic tails of the receptors. The TGN is the site where newly synthesized lysosomal enzymes bind to MPRs that are then collected into clathrin-coated pits and budded into clathrin-coated vesicles for delivery to the early endosome. This process involves interaction of the MPRs with two types of coat proteins: the GGAs (Golgi-localized, γ-ear-containing, ADP-ribosylation factor binding) and AP1 (adapter protein 1). In addition to binding MPRs, the coat proteins recruit clathrin for the assembly of clathrin-coated vesicles. Following delivery to early endosomes, lysosomal enzymes are released from MPRs as the endosomes mature to late endosomes and the pH decreases. Late endosomes then undergo dynamic fusion/fission with lysosomes, allowing selective transfer of lysosomal enzymes to the lysosomes and leaving the MPRs behind in subdomains of the late endosomes. These MPRs may then either return to the TGN or move to the plasma membrane, where internalization via clathrin-coated pits occurs, mediated by the coat protein AP2. There are several pathways for the MPRs to be returned to the TGN from the various endosomal compartments, although the relative importance of the different pathways is unclear at this time.

FIGURE 30.5

Subcellular trafficking pathways followed by glycoproteins, lysosomal enzymes, and MPRs. Newly synthesized glycoproteins originating from the rough ER pass through the Golgi stacks and are then sorted to various destinations as indicated. Along this route, (more...)

RELATIVE ROLES OF THE TWO MPRS IN INTRACELLULAR TRAFFICKING

In many cell types, a minority of newly synthesized lysosomal enzyme molecules escape sorting and are secreted into the medium even though they carry M6P residues (Figure 30.5). Such secreted molecules may be recaptured by the same cell or by adjacent cells expressing cell-surface MPRs. Enzyme molecules that bind to such cell-surface MPRs are endocytosed via clathrin-coated pits and vesicles, eventually reaching the same late endosomal compartments where newly synthesized molecules arrive from the Golgi. This “secretion-recapture” pathway is a minor one in most cells, but has potential importance in some situations. For example, some activated macrophages secrete a large portion of their lysosomal enzymes directly into the medium. It is possible that under inflammatory situations, it is useful for such secreted enzymes to be returned to lysosomes via the MPR pathway.

As indicated above, cells genetically deficient in both receptors secrete most of their enzymes, much like cells from patients with I-cell disease. Under normal conditions, only the CI-MPR is responsible for lysosomal enzyme endocytosis from the cell surface. However, when the CD-MPR is strongly overexpressed, it is capable of mediating uptake from the plasma membrane. These differences may be due to the narrow pH optimum of the CD-MPR for binding ligand and/or its variable oligomeric state. Taken together, the results indicate that although the CI-MPR is a major determinant of trafficking in the biosynthetic pathway, the CD-MPR also contributes significantly. Curiously, when the CD-MPR is overexpressed in cells containing the CI-MPR, increased secretion of lysosomal enzymes can result. Thus, the CD-MPR may modulate the pathway in the direction of either retention or secretion, perhaps based on factors such as its oligomeric state, expression level, subcompartmental pH, divalent cation availability, amounts of CI-MPR present, and/or differences in precise affinities for multivalent ligands (Table 30.1). In the final analysis, different combinations of amounts and locations of the two receptors, together with the spectrum of M6P recognition markers on various enzymes, could explain the highly variable physiology of lysosomal enzyme trafficking in different cell types.

TABLE 30.1

Comparison of the two mammalian M6P receptors (P-type lectins)

IMPLICATIONS FOR ENZYME REPLACEMENT THERAPY

As described in Chapter 41, there are many genetic disorders in glycan degradation that result from decreased activity of a given lysosomal enzyme. Some of these enzymes that are targeted to lysosomes via the M6P pathway have been prepared in large quantities as recombinant soluble proteins and used in enzyme replacement therapy. To date the benefits have been variable but less than optimal. There are a number of potential reasons for this. First, some of the preparations do not contain the physiologic complement of the phosphomannosyl recognition marker. It is reasonable to suggest that the M6P-MPR system might be used to improve the efficacy of enzyme replacement in these patients. This has been shown to be the case in mouse and dog model systems. However, even with fully phosphorylated enzymes there may be obstacles that are difficult to overcome. For example, some cell types in the body may not express adequate levels of the CI-MPR on their surfaces to endocytose sufficient enzyme to restore normal lysosomal function. Also, the organ that is most seriously affected in many of these diseases (the brain) is inaccessible because of the blood–brain barrier. This is an area where further studies are needed, and replacement therapy using the MPR system might end up being more effective in selected circumstances.

EVOLUTIONARY ORIGINS OF THE M6P RECOGNITION SYSTEM

Although the MPR pathway has a major role in vertebrate lysosomal enzyme trafficking, its contribution in invertebrate systems is not prominent. Lysosomal enzymes are targeted in organisms such as Saccharomyces, Trypanosoma, and Dictyostelium, without the aid of identifiable MPRs. The slime mold Dictyostelium discoideum produces a novel methylphosphomannose structure on some of its lysosomal enzymes that can be recognized in vitro by the mammalian CI-MPR (not the CD-MPR). However, despite the presence of a GlcNAc-P-T that recognizes α1-2-linked mannose residues, the enzyme does not specifically recognize lysosomal enzymes, and no receptor for M6P has been found in this organism. The protozoan Acanthamoeba produces a GlcNAc-P-T that specifically recognizes lysosomal enzymes. However, this organism lacks a gene that could encode an “uncovering” enzyme, and so would not be expected to form M6P available to an MPR. Although some additional “lower” organisms do show evidence for an “uncovering” enzyme, no MPR activity has yet been found. The evolutionary divergence point at which the complete MPR system became established remains to be defined.

THE CATION-INDEPENDENT RECEPTOR BINDS MANY OTHER LIGANDS

Although originally discovered as a receptor for lysosomal enzyme trafficking, the CI-MPR turns out to be a remarkably multifunctional molecule. IGF-II was previously known to bind two receptors, one identical to the IGF-I receptor and another independent receptor. Molecular cloning of the latter receptor revealed the surprising fact that it is identical to the CI-MPR. Importantly, IGF-II does not carry M6P residues. Many studies have since explored potential interactions between these disparate ligands for the CI-MPR. Although the two ligands bind to distinct sites on the receptor, there are conflicting reports regarding synergistic or antagonistic interactions between the two activities. It has also been suggested that the redistribution of the CI-MPR upon IGF-II stimulation could explain some of the known metabolic effects of this hormone on protein degradation, by altering the trafficking of lysosomal enzymes. However, CI-MPRs of the chicken and Xenopus do not bind IGF-II, although their cells can respond to IGF-II. This makes it less likely that the overlap in binding specificity is of vital importance to animal cells in general. Rather, it appears that the CI-MPR acts primarily as a general “sink” for excess IGF-II in the extra-cellular fluid, carrying it to the lysosome for degradation, and reducing the amount available to bind to the IGF-I receptor. A number of reports indicate that binding of IGF-II to the CI-MPR regulates motility and growth in some cell types. It has also been found that the CI-MPR binds retinoic acid with high affinity at a site that is distinct from those for M6P and IGF-II. This binding of retinoic acid seems to enhance the primary functions of the CI-MPR receptor, and the biological consequence appears to be the suppression of cell proliferation and/or induction of apoptosis. The significance of this unexpected observation is still being explored. Other interesting ligands include urokinase-type plasminogen activator receptor (uPAR) and plasminogen.

There are also some unexplained changes in CI-MPR expression in relation to malignancy. Loss of heterozygosity at the CI-MPR locus occurs in dysplastic liver lesions and in hepatocellular carcinomas associated with the high-risk factors of hepatitis virus infection and liver cirrhosis. Mutations in the remaining allele were detected in about 50% of these tumors, which also seem to frequently develop from clonal expansions of phenotypically normal, CI-MPR-mutated hepatocytes. Thus, the CI-MPR fulfills many of the classic criteria to be classified as a liver “tumor-suppressor” gene.

SIGNIFICANCE OF M6P ON NONLYSOSOMAL PROTEINS

Interestingly, M6P-containing N-glycans have been found on a variety of nonlysosomal proteins. Some are hydrolytic enzymes that seem to have taken on a predominantly secretory route, for example, uteroferrin and DNase I. In the first case, the failure of removal of the blocking N-acetylglucosamine residues may be the cause for secretion. With DNase I, the native level of phosphorylation simply appears to be low. M6P has been found on the transforming growth factor β (TGF-β) precursor and the phosphate is lost from the mature form. It appears that M6P may serve to target the precursor to CI-MPR for activation. Other nonlysosomal proteins reported to carry M6P include proliferin, CREG (cellular repressor of E1A-stimulated genes), LIF (leukemia inhibitory factor), and thyroglobulin. In the last case, M6P-containing N-glycans are suggested as a mechanism to target the protein for degradation and release of thyroid hormones. One should not necessarily assume that M6P-containing N-glycans on all of these proteins are involved in intracellular trafficking. Just as phosphorylation of serine residues has diverse biological roles, M6P might be used for more than one purpose in a complex multicellular organism. Further investigation of each situation is therefore needed, with an open mind to all of the possibilities.

Herpes simplex virus and varicella zoster virus (VZV) glycoproteins have also been shown to carry N-glycans with M6P. In these cases, M6P is on complex N-glycans, suggesting that it originates from a distinct biosynthetic pathway. Regardless of its mode of synthesis, interaction of cell-free VZV with CI-MPR at the cell surface is required for viral entry into endosomes. Interestingly, intracellular CI-MPR can also divert newly synthesized enveloped VZV to late endosomes where the virions are inactivated before exocytosis. This is suggested as the mechanism by which this successful parasite limits immediate excessive spread and avoids killing the host. Biopsies of VZV-infected human skin showed that CI-MPR expression is lost in maturing superficial epidermal cells, preventing diversion of VZV to endosomes and allowing constitutive secretion of infectious VZV. These data implicate CI-MPR in the complex biology of VZV infection.

OTHER PATHWAYS FOR TRAFFICKING LYSOSOMAL ENZYMES

Although the M6P recognition marker has a crucial role in trafficking newly synthesized lysosomal enzymes to vertebrate lysosomes, alternate mechanisms evidently exist in some cell types. Even in I-cell disease, some cells and tissues (e.g., liver and circulating granulocytes) have essentially normal levels of enzymes. B-lymphoblast lines derived from these patients also do not show the complete phenotype of enzyme deficiency seen in fibroblasts. One interpretation is that the M6P pathway for trafficking of lysosomal enzymes is a specialized form of targeting, superimposed on some other basic mechanisms that evolutionarily more primitive organisms use, some of which remain undefined. In this regard, two lysosomal enzymes, acid phosphatase and β-glucocerebrosidase, are not at all affected in their distribution in I-cell disease fibroblasts. With acid phosphatase, the enzyme is synthesized initially as a membrane-bound protein, and once in the lysosome, it is proteolytically cleaved to generate the mature soluble form. Glucocerebrosidase is also membrane associated, does not show phosphorylation of its glycans, and is targeted to lysosomes independently of the MPR pathway. Likewise, integral membrane proteins of the lysosome such as the LAMP/lgp proteins do not require the M6P recognition marker pathway for trafficking to lysosomes. Rather, they seem to use motifs in their cytoplasmic tails similar to those that target MPRs to clathrin-coated vesicles.

FURTHER READING

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