MAL mediates apical transport of secretory proteins in polarized epithelial Madin-Darby canine kidney cells.

The MAL proteolipid is an integral membrane protein identified as a component of the raft machinery for apical sorting of membrane proteins in Madin-Darby canine kidney (MDCK) cells. Previous studies have implicated lipid rafts in the transport of exogenous thyroglobulin (Tg), the predominant secretory protein of thyroid epithelial cells, to the apical surface in MDCK cells. We have examined the secretion of recombinant Tg and gp80/clusterin, a major endogenous secretory protein not detected in Triton X-100 insoluble rafts, for the investigation of the involvement of MAL in the constitutive apical secretory pathway of MDCK cells. We show that MAL depletion impairs apical secretion of Tg and causes its accumulation in the Golgi. Cholesterol sequestration, which blocks apical secretion of Tg, did not alter the levels of MAL in rafts but created a block proximal to Tg entrance into rafts. Apical secretion of gp80/clusterin was also inhibited by elimination of endogenous MAL. Our results suggest a role for MAL in the transport of both endogenously and exogenously expressed apical secretory proteins in MDCK cells.     

Identification and function of disulfide bridges in the extracellular domains of the angiotensin II type 2 receptor.

The angiotensin II (AngII) receptor family is comprised of two subtypes, type 1 (AT(1)) and type 2 (AT(2)). Although sharing low homology (only 34%), mutagenesis has identified some key residues that are conserved between both subtypes, including four extracellular cysteines. Previous AT(1) mutagenesis demonstrated that the cysteines form two disulfide bonds, one linking the first and second extracellular loops and another connecting the amino terminus to the third extracellular loop. The importance of these AT(1) disulfides in ligand binding is supported by the effect of dithiothreitol (DTT). DTT breaks disulfide bonds, thereby strongly inhibiting ligand binding in AT(1) receptors. Despite retaining the same cysteines, AT(2) receptor ligand binding is paradoxically enhanced by DTT. Thus, we constructed a series of AT(2) cysteine mutations, either individually or paired, to establish the role of the cysteines and the source of DTT's effects. The AT(2) cysteine mutants surprisingly confirmed that the cysteines form disulfide bonds in the same manner as in the AT(1) subtype. However, breaking the AT(2) disulfide bridges yielded two responses. As in AT(1) receptors, mutations disrupting the disulfide bond between the first and second extracellular loops reduced AT(2) binding by 4-fold. In contrast, mutations breaking the disulfide bridge between the amino terminus and the third extracellular loop increased AT(2) binding, mimicking DTT's effect on this subtype. Further analysis of AT(1)/AT(2) chimeric exchange mutants of these domains suggested that the AT(2) amino terminus and third extracellular loop may possess latent binding epitopes that are only uncovered after DTT exposure.     

The Cholinesterase-like Domain, Essential in Thyroglobulin Trafficking for Thyroid Hormone Synthesis, Is Required for Protein Dimerization

The carboxyl-terminal cholinesterase-like (ChEL) domain of thyroglobulin (Tg) has been identified as critically important in Tg export from the endoplasmic reticulum. In a number of human kindreds suffering from congenital hypothyroidism, and in the cog congenital goiter mouse and rdw rat dwarf models, thyroid hormone synthesis is inhibited because of mutations in the ChEL domain that block protein export from the endoplasmic reticulum. We hypothesize that Tg forms homodimers through noncovalent interactions involving two predicted α-helices in each ChEL domain that are homologous to the dimerization helices of acetylcholinesterase. This has been explored through selective epitope tagging of dimerization partners and by inserting an extra, unpaired Cys residue to create an opportunity for intermolecular disulfide pairing. We show that the ChEL domain is necessary and sufficient for Tg dimerization; specifically, the isolated ChEL domain can dimerize with full-length Tg or with itself. Insertion of an N-linked glycan into the putative upstream dimerization helix inhibits homodimerization of the isolated ChEL domain. However, interestingly, co-expression of upstream Tg domains, either in cis or in trans, overrides the dimerization defect of such a mutant. Thus, although the ChEL domain provides a nidus for Tg dimerization, interactions of upstream Tg regions with the ChEL domain actively stabilizes the Tg dimer complex for intracellular transport.The synthesis of thyroid hormone in the thyroid gland requires secretion of thyroglobulin (Tg)² to the apical luminal cavity of thyroid follicles (1). Once secreted, Tg is iodinated via the activity of thyroid peroxidase (2). A coupling reaction involving a quinol-ether linkage especially engages di-iodinated tyrosyl residues 5 and 130 to form thyroxine within the amino-terminal portion of the Tg polypeptide (3, 4). Preferential iodination of Tg hormonogenic sites is dependent not on the specificity of the peroxidase (5) but upon the native structure of Tg (6, 7). To date, no other thyroidal proteins have been shown to effectively substitute in this role for Tg.The first 80% of the primary structure of Tg (full-length murine Tg: 2,746 amino acids) involves three regions called I-II-III comprised of disulfide-rich repeat domains held together by intradomain disulfide bonds (8, 9). The final 581 amino acids of Tg are strongly homologous to acetylcholinesterase (10–12). Rate-limiting steps in the overall process of Tg secretion involve its structural maturation within the endoplasmic reticulum (ER) (13). Interactions between regions I-II-III and the cholinesterase-like (ChEL) domain have recently been suggested to be important in this process, with ChEL functioning as an intramolecular chaperone and escort for I-II-III (14). In addition, Tg conformational maturation culminates in Tg homodimerization (15, 16) with progression to a cylindrical, and ultimately, a compact ovoid structure (17–19).In human congenital hypothyroidism with deficient Tg, the ChEL domain is a commonly affected site of mutation, including the recently described A2215D (20, 21), R2223H (22), G2300D, R2317Q (23), G2355V, G2356R, and the skipping of exon 45 (which normally encodes 36 amino acids), as well as the Q2638stop mutant (24) (in addition to polymorphisms including P2213L, W2482R, and R2511Q that may be associated with thyroid overgrowth (25)). As best as is currently known, all of the congenital hypothyroidism-inducing Tg mutants are defective for intracellular transport (26). A homozygous G2300R mutation (equivalent to residue 2,298 of mouse Tg) in the ChEL domain is responsible for congenital hypothyroidism in rdw rats (27, 28), whereas we identified the Tg-L2263P point mutation as the cause of hypothyroidism in the cog mouse (29). Such mutations perturb intradomain structure (30), and interestingly, block homodimerization (31). Acquisition of quaternary structure has long been thought to be required for efficient export from the ER (32) as exemplified by authentic acetylcholinesterase (33, 34) in which dimerization enhances protein stability and export (35).Tg comprised only of regions I-II-III (truncated to lack the ChEL domain) is blocked within the ER (30), whereas a secretory version of the isolated ChEL domain of Tg devoid of I-II-III undergoes rapid and efficient intracellular transport and secretion (14). A striking homology positions two predicted α-helices of the ChEL domain to the identical relative positions of the dimerization helices in acetylcholinesterase. This raises the possibility that ChEL may serve as a homodimerization domain for Tg, providing a critical function in maturation for Tg transport to the site of thyroid hormone synthesis (1).In this study, we provide unequivocal evidence for homodimerization of the ChEL domain and “hetero”-dimerization of that domain with full-length Tg, and we provide significant evidence that the predicted ChEL dimerization helices provide a nidus for Tg assembly. On the other hand, our data also suggest that upstream Tg regions known to interact with ChEL (14) actively stabilize the Tg dimer complex. Together, I-II-III and ChEL provide unique contributions to the process of intracellular transport of Tg through the secretory pathway.     

A conserved histidine in insulin is required for the foldability of human proinsulin: structure and function of an ALAB5 analog

The insulins of eutherian mammals contain histidines at positions B5 and B10. The role of His(B10) is well defined: although not required in the mature hormone for receptor binding, in the islet beta cell this side chain functions in targeting proinsulin to glucose-regulated secretory granules and provides axial zincbinding sites in storage hexamers. In contrast, the role of His(B5) is less well understood. Here, we demonstrate that its substitution with Ala markedly impairs insulin chain combination in vitro and blocks the folding and secretion of human proinsulin in a transfected mammalian cell line. The structure and stability of an Ala(B5)-insulin analog were investigated in an engineered monomer (DKP-insulin). Despite its impaired foldability, the structure of the Ala(B5) analog retains a native-like T-state conformation. At the site of substitution, interchain nuclear Overhauser effects are observed between the methyl resonance of Ala(B5) and side chains in the A chain; these nuclear Overhauser effects resemble those characteristic of His(B5) in native insulin. Substantial receptor binding activity is retained (80 +/- 10% relative to the parent monomer). Although the thermodynamic stability of the Ala(B5) analog is decreased (DeltaDeltaG(u) = 1.7 +/- 0.1 kcal/mol), consistent with loss of His(B5)-related interchain packing and hydrogen bonds, control studies suggest that this decrement cannot account for its impaired foldability. We propose that nascent long-range interactions by His(B5) facilitate alignment of Cys(A7) and Cys(B7) in protein-folding intermediates; its conservation thus reflects mechanisms of oxidative folding rather than structure-function relationships in the native state.     

Mutations in the arylsulfatase A pseudodeficiency allele causing metachromatic leukodystrophy.

We identified a patient suffering from late infantile metachromatic leukodystrophy who genetically seemed to be homozygous for the mutations signifying the arylsulfatase A pseudodeficiency allele. Homozygosity for the pseudodeficiency allele is associated with low arylsulfatase A activity but does not cause a disease. Analysis of the arylsulfatase A gene in this patient revealed a C----T transition in exon 2, causing a Ser 96----Phe substitution in addition to the sequence alterations causing arylsulfatase A pseudodeficiency. Although this mutation was found only in 1 of 78 metachromatic leukodystrophy patients tested, five more patients were identified who seemed hetero- or homozygous for the pseudodeficiency allele. The existence of nonfunctional arylsulfatase A alleles derived from the pseudodeficiency allele calls for caution when the diagnosis of arylsulfatase A pseudodeficiency is based solely on the identification of the mutations characterizing the pseudodeficiency allele.     

Two new arylsulfatase A (ARSA) mutations in a juvenile metachromatic leukodystrophy (MLD) patient.

Fragments of the arylsulfatase A (ARSA) gene from a patient with juvenile-onset metachromatic leukodystrophy (MLD) were amplified by PCR and ligated into MP13 cloning vectors. Clones hybridizing with cDNA for human ARSA were selected, examined for appropriate size inserts, and used to prepare single-stranded phage DNA. Examination of the entire coding and most of the intronic sequence revealed two putative disease-related mutations. One, a point mutation in exon 3, resulted in the substitution of isoleucine by serine. Introduction of this alteration into the normal ARSA cDNA sequence resulted in a substantial decrease in ARSA activity on transient expression in cultured baby hamster kidney cells. About 5% of the control expression was observed, suggesting a small residual activity in the mutated ARSA. The second mutation, a G-to-A transition, occurred in the other allele and resulted in an altered splice-recognition sequence between exon 7 and the following intron. The mutation also resulted in the loss of a restriction site. Apparently normal levels of mRNA were generated from this allele, but no ARSA activity or immuno-cross-reactive material could be detected. A collection of DNA samples from known or suspected MLD patients, members of their families, and normal controls was screened for these mutations. Four additional individuals carrying each of the mutations were found among the nearly 100 MLD patients in the sample. Gene segregation in the original patient's family was consistent with available clinical and biochemical data. No individuals homozygous for either of these two mutations were identified. However, combinations with other MLD mutations suggest that the point mutation in exon 3 does result in some residual enzyme activity and is associated with late-onset forms of the disease. The splice-site mutation following exon 7 produces late-infantile MLD when combined with other enzyme-null mutations, implying that it is completely silent enzymatically.     

Crystal Structure of a ��Nonfoldable�� Insulin: IMPAIRED FOLDING EFFICIENCY DESPITE NATIVE ACTIVITY*

Protein evolution is constrained by folding efficiency (“foldability”) and the implicit threat of toxic misfolding. A model is provided by proinsulin, whose misfolding is associated with β-cell dysfunction and diabetes mellitus. An insulin analogue containing a subtle core substitution (Leu^A16 → Val) is biologically active, and its crystal structure recapitulates that of the wild-type protein. As a seeming paradox, however, Val^A16 blocks both insulin chain combination and the in vitro refolding of proinsulin. Disulfide pairing in mammalian cell culture is likewise inefficient, leading to misfolding, endoplasmic reticular stress, and proteosome-mediated degradation. Val^A16 destabilizes the native state and so presumably perturbs a partial fold that directs initial disulfide pairing. Substitutions elsewhere in the core similarly destabilize the native state but, unlike Val^A16, preserve folding efficiency. We propose that Leu^A16 stabilizes nonlocal interactions between nascent α-helices in the A- and B-domains to facilitate initial pairing of Cys^A20 and Cys^B19, thus surmounting their wide separation in sequence. Although Val^A16 is likely to destabilize this proto-core, its structural effects are mitigated once folding is achieved. Classical studies of insulin chain combination in vitro have illuminated the impact of off-pathway reactions on the efficiency of native disulfide pairing. The capability of a polypeptide sequence to fold within the endoplasmic reticulum may likewise be influenced by kinetic or thermodynamic partitioning among on- and off-pathway disulfide intermediates. The properties of [Val^A16]insulin and [Val^A16]proinsulin demonstrate that essential contributions of conserved residues to folding may be inapparent once the native state is achieved.