The glycan domain of thrombopoietin (TPO) acts in trans to enhance secretion of the hormone and other cytokines

Thrombopoietin (TPO), the primary regulator of platelet production, is composed of an amino-terminal 152 amino acids, sufficient for activity, and a carboxyl-terminal region rich in carbohydrates (183 residues) that enhances secretion of the molecule. Full-length TPO is secreted at levels 10-20-fold greater than truncated TPO. By introducing into mammalian cells a novel cDNA encoding the TPO secretory leader linked to its carboxyl-terminal domain (TPO glycan domain (TGD)), we tested whether TGD could function in trans to enhance secretion of TPO. The artificial TGD was secreted, inactive in proliferation assays, and did not inhibit TPO activity. However, when co-transfected with a cDNA encoding truncated TPO, TGD enhanced secretion 4-fold, measured by specific bioassay and immunoassay. TGD also enhanced secretion of granulocyte monocyte colony-stimulating factor and stem cell factor but did not affect the production of erythropoietin, interleukin-3, growth hormone, or of full-length TPO. To localize TGD function, we added an endoplasmic reticulum (ER) retention signal to TGD and, separately, deleted the secretory leader. Deletion of the secretory leader attenuated the secretory function of TGD, whereas addition of the ER retention signal did not alter its function. To investigate the physiologic role of TGD in folding and proteasomal protection, we tested full-length and truncated TPO in assays of protein refolding, and we examined protein stability in the presence of proteasome inhibitors. We found that truncated TGD re-folds readily and that proteasome-mediated degradation contributes to the poor secretion of truncated TPO. We conclude that TGD enhances secretion of TPO and can additionally function as an inter-molecular chaperone, in part because of its ability to prevent degradation of the hormone. The cellular location of TGD action is likely to be within the ER or earlier in the secretory pathway.     

Cloning and sequencing of a human pancreatic tumor mucin cDNA

A monospecific polyclonal antiserum against deglycosylated human pancreatic tumor mucin was used to select human pancreatic mucin cDNA clones from a lambda gt11 cDNA expression library developed from a human pancreatic tumor cell line. The full-length 4.4-kilobase mucin cDNA sequence included a 72-base pair 5'-untranslated region and a 307-base pair 3'-untranslated region. The predicted amino acid sequence for this cDNA revealed a protein of 122,071 daltons containing 1,255 amino acid residues of which greater than 60% were serine, threonine, proline, alanine, and glycine. Approximately two-thirds of the protein sequence consisted of identical 20-amino acid tandem repeats which were flanked by degenerate tandem repeats and nontandem repeat sequences on both the amino-terminal and carboxyl-terminal ends. The amino acid sequence also contained five putative N-linked glycosylation sites, a putative signal sequence and transmembrane domain, and numerous serine and threonine residues (potential O-linked glycosylation sites) outside and within the tandem repeat position. The cDNA and deduced amino acid sequence of the pancreatic mucin sequence was over 99% homologous with a mucin cDNA sequence derived from breast tumor mucin, even though the native forms of these molecules are quite distinct in size and degree of glycosylation.     

Characterization of the carboxyl-terminal domain of the rat glucose-dependent insulinotropic polypeptide (GIP) receptor. A role for serines 426 and 427 in regulating the rate of internalization.

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone involved in the regulation of insulin secretion. In non-insulin-dependent diabetes mellitus insulin responses to GIP are blunted, possibly due to altered signal transduction or reduced receptor number. Site-directed mutagenesis was used to construct truncated GIP receptors to study the importance of the carboxyl-terminal tail (CT) in binding, signaling, and receptor internalization. Receptors truncated at amino acids 425, 418, and 405, expressed in COS-7 or CHO-K1 cells, exhibited similar binding to wild type receptors. GIP-dependent cAMP production with the 405 mutant was decreased in COS-7 cells. Maximal cAMP production in CHO-K1 cells was reduced with all truncated forms. Binding was undetectable with a receptor truncated at amino acid 400; increasing tail length by adding 5 alanines restored binding and signaling. Mutants produced by alanine scanning of residues 394-401, adjacent to transmembrane domain 7, were all functional. CT truncation by 30 or more amino acids, mutation of serines 426/427, singly or combined, or complete CT serine knockout all reduced receptor internalization rate. The majority of the GIP receptor CT is therefore not required for signaling, a minimum chain length of approximately 405 amino acids is needed for receptor expression, and serines 426 and 427 are important for regulating rate of receptor internalization.     

Affinity-defining domains in the Na-Cl cotransporter: a different location for Cl- and thiazide binding

The thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the distal convoluted tubule, serves as a receptor for thiazide-type diuretics, and is involved in inherited diseases associated with abnormal blood pressure. Little is known regarding the structure-function relationship in this cotransporter. Previous studies from our group reveal that mammalian NCC exhibits higher affinity for ions and thiazides than teleost NCC and suggest a role for glycosylation upon thiazide affinity. Here we have constructed a series of chimeric and mutant cDNAs between rat and flounder NCC to define the role of glycosylation status, the amino-terminal domain, the carboxyl-terminal domain, the extracellular glycosylated loop, and the transmembrane segments upon affinity for Na+, Cl-, and metolazone. Xenopus laevis oocytes were used as the heterologous expression system. We observed that elimination of glycosylation sites in flounder NCC did not affect the affinity of the cotransporter for metolazone. Also, swapping the amino-terminal domain, the carboxyl-terminal domain, the glycosylation sites, or the entire extracellular glycosylation loop between rat and flounder NCC had no effect upon ions or metolazone affinity. In contrast, interchanging transmembrane regions between rat and flounder NCC revealed that affinity-modifying residues for chloride are located within the transmembrane 1-7 region and for thiazides are located within the transmembrane 8-12 region, whereas both segments seem to be implicated in defining sodium affinity. These observations strongly suggest that binding sites for chloride and thiazide in NCC are different.     

Essential hydrophilic carboxyl-terminal regions including cysteine residues of the yeast stretch-activated calcium-permeable channel Mid1.

The yeast Saccharomyces cerevisiae MID1 gene encodes a stretch-activated Ca(2+)-permeable nonselective cation channel composed of 548 amino acid residues. A physiological role of the Mid1 channel is known to maintain the viability of yeast cells exposed to mating pheromone, but its structural basis remains to be clarified. To solve this problem, we identified the mutation sites of mid1 mutant alleles generated by in vivo ethyl methanesulfonate mutagenesis and found that two mid1 alleles have nonsense mutations at the codon for Trp(441), generating a truncated Mid1 protein lacking two-thirds of the intracellular carboxyl-terminal region from Asn(389) to Thr(548). In vitro random mutagenesis with hydroxylamine also showed that the carboxyl-terminal region is essential. To identify the functional portion of the carboxyl-terminal region in detail, we performed a progressive carboxyl-terminal truncation followed by functional analyses and found that the truncated protein produced from the mid1 allele bearing the amber mutation at the codon for Phe(522) (F522Am) complemented the mating pheromone-induced death phenotype of the mid1 mutant and increased its Ca(2+) uptake activity to a wild-type level, whereas N521Am did not. This result indicates that the carboxyl-terminal domain spanning from Asn(389) to Asn(521) is required for Mid1 function. Interestingly, this domain is cysteine-rich, and alanine-scanning mutagenesis revealed that seven out of 10 cysteine residues are unexchangeable. These results clearly indicate that the carboxyl-terminal domain including the cysteine residues is important for Mid1 function.     

Multiple sites of contact between the carboxyl-terminal binding domain of PTHrP-(1--36) analogs and the amino-terminal extracellular domain of the PTH/PTHrP receptor identified by photoaffinity cross-linking

The carboxyl-terminal portions of parathyroid hormone (PTH)-(1--34) and PTH-related peptide (PTHrP)-(1-36) are critical for high affinity binding to the PTH/PTHrP receptor (P1R), but the mechanism of receptor interaction for this domain is largely unknown. To identify interaction sites between the carboxyl-terminal region of PTHrP-(1--36) and the P1R, we prepared analogs of [I(5),W(23),Y(36)]PTHrP-(1--36)-amide with individual p-benzoyl-l-phenylalanine (Bpa) substitutions at positions 22--35. When tested with LLC-PK(1) cells stably transfected with human P1R (hP1R), the apparent binding affinity and the EC(50) of agonist-stimulated cAMP accumulation for each analog was, with the exception of the Bpa(24)-substituted analog, similar to that of the parent compound. The radiolabeled Bpa(23)-, Bpa(27)-, Bpa(28)-, and Bpa(33)-substituted compounds affinity-labeled the hP1R sufficiently well to permit subsequent mapping of the cross-linked receptor region. Each of these peptides cross-linked to the amino-terminal extracellular domain of the P1R: [I(5),Bpa(23),Y(36)]PTHrP-(1-36)-amide cross-linked to the extreme end of this domain (residues 33-63); [I(5),W(23),Bpa(27),Y(36)]PTHrP-(1--36)-amide cross-linked to residues 96--102; [I(5),W(23),Bpa(28),Y(36)]PTHrP-(1--36)- amide cross-linked to residues 64--95; and [I(5),W(23), Bpa(33),Y(36)]PTHrP-(1--36)-amide cross-linked to residues 151-172. These data thus predict that residues 23, 27, 28, and 33 of native PTHrP are each near to different regions of the amino-terminal extracellular receptor domain of the P1R. This information helps define sites of proximity between several ligand residues and this large receptor domain, which so far has been largely excluded from models of the hormone-receptor complex.     

Isolation and characterization of two biologically active O-glycosylated forms of human parathyroid hormone produced in Saccharomyces cerevisiae. Identification of a new motif for O-glycosylation.

Expression and secretion of human parathyroid hormone in Saccharomyces cerevisiae were achieved by fusing a cDNA encoding the mature human parathyroid hormone (hPTH) to the preproregion of the yeast mating factor alpha. Purified hPTH from yeast-culture medium was found to contain, in addition to the native unglycosylated form, two mannosylated variants with different molecular masses. The three hPTH forms were processed identically, resulting in the same 84 amino acid polypeptides with amino acid sequences identical to the native hormone. In both the O-glycosylated forms that were separated by isocratic reverse-phase HPLC, two mannose-linked residues were localized to Thr79. In addition, the most glycosylated form showed a heterogeneous modification of three, four or five mannosyl residues linked at Ser66. Lysine is N-terminally located to Ser66 and probably stimulates this glycosylation, which introduces a possible new motif for O-glycosylation in yeast. The two glycosylated forms of hPTH had similar biological activity which was identical to the native form of hPTH in a hormone-sensitive adenylate cyclase assay in bone sarcoma cells. Thus, a C-terminal O-glycosylation of hPTH with up to seven mannosyl residues/molecule did not affect the biological activity of the hormone, making possible production of hPTH with potential different pharmacokinetic properties.     

Oligomerization, chaperone activity, and nuclear localization of p26, a small heat shock protein from Artemia franciscana

Artemia franciscana embryos undergo encystment, developmental arrest and diapause, the last characterized by profound metabolic dormancy and extreme stress resistance. Encysted embryos contain an abundant small heat shock protein termed p26, a molecular chaperone that undoubtedly has an important role in development. To understand better the role of p26 in Artemia embryos, the structural and functional characteristics of full-length and truncated p26 expressed in Escherichia coli and COS-1 cells were determined. p26 chaperone activity declined with increasing truncation of the protein, and those deletions with the greatest adverse effect on protection of citrate synthase during thermal stress had the most influence on oligomerization. When produced in either prokaryotic or eukaryotic cells the p26 alpha-crystallin domain consisting of amino acid residues 61-152 existed predominantly as monomers, and p26 variants lacking the amino-terminal domain but with intact carboxyl-terminal extensions were mainly monomers and dimers. The amino terminus was, therefore, required for efficient dimer formation. Assembly of higher order oligomers was enhanced by the carboxyl-terminal extension, although removing the 10 carboxyl-terminal residues had relatively little effect on oligomerization and chaperoning. Full-length and carboxyl-terminal truncated p26 resided in the cytoplasm of transfected COS-1 cells; however, variants missing the complete amino-terminal domain and existing predominantly as monomers/dimers entered the nuclei. A mechanism whereby oligomer disassembly assisted entry of p26 into nuclei was suggested, this of importance because p26 translocates into Artemia embryo nuclei during development and stress. However, when examined in Artemia, the p26 oligomer size was unchanged under conditions that allowed movement into nuclei, suggesting a process more complex than just oligomer dissociation.     

Isolation and sequencing of a cDNA clone encoding lysosomal membrane glycoprotein mouse LAMP-1. Sequence similarity to proteins bearing onco-differentiation antigens

We have isolated and sequenced a cDNA clone encoding the mouse LAMP-1 (mLAMP-1) major lysosomal membrane glycoprotein. The deduced protein sequence, which included the NH2-terminal portion of the mLAMP-1 molecule, consisted of 382 amino acids (Mr 41,509). The predicted structure of this protein included an NH2-terminal intralumenal domain consisting of two homology units of approximately 160 residues each separated by a proline-rich hinge region. Each homology unit contained four cysteine residues with two intercysteine intervals of 36-38 residues and one of 68 or 76 residues. The molecule also contained 20 asparagine-linked glycosylation sites within residues 1-287, a membrane-spanning region from residues 347 to 370, and a carboxyl-terminal cytoplasmic domain of 12 residues. The biochemical properties and amino acid sequence of mLAMP-1 were highly similar to those of two other molecules that have been studied as cell surface onco-differentiation antigens: a highly sialylated polylactosaminoglycan-containing glycoprotein isolated from human chronic myelogenous leukemia cells (Viitala, J., Carlsson, S. R., Siebert, P. D., and Fukuda, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, in press) and the mouse gp130 (P2B) glycoprotein, in which an increase in beta 1-6 branching of asparagine-linked oligosaccharides has been correlated with metastatic potential in certain tumor cells (Dennis, J.W., Laferte, S., Waghorne, C., Breitman, M.L., and Kerbel, R.S. (1987) Science 236, 582-585).     

Functional analysis of the C-terminal region of recombinant human thrombopoietin. C-terminal region of thrombopoietin is a "shuttle" peptide to help secretion

Thrombopoietin (TPO) is a cytokine that primarily stimulates megakaryocytopoiesis and thrombopoiesis. TPO has a unique C-terminal tail peptide of about 160 amino acids that consists mostly of hydrophilic residues and contains six N-linked sugar chains. In order to investigate the biological function of the C-terminal domain, two series of mutations were performed. One is systematic truncation from the C terminus. Another is elimination of N-glycosylation sites in the C-terminal domain by Asn to Gln mutations. After the mutant proteins were expressed by mammalian cells, it was found that the elimination of the N-linked sugar sites did not affect the biological activity, whereas truncation of the C-terminal domain resulted in elevation of in vitro activity up to 4-fold. The C-terminal peptide itself was found to inhibit the in vitro activity. Moreover, both the C-terminal truncation and the elimination of the N-glycosylation sites decreased the secretion level progressively down to (1)/(10) that of wild type, and the amount of the mutant left in the cell increased. The N-glycosylation in the C-terminal region was found to be important for secretion of TPO. Among six N-glycosylation sites in the C-terminal region, two locations, Asn-213 and Asn-234, were found to be critical for secretion, and two other locations, Asn-319 and Asn-327, did not affect the secretion.     

Glycosylation of human apolipoprotein E. The carbohydrate attachment site is threonine 194

The glycosylation of human apolipoprotein (apo) E was examined with purified plasma apoE and apoE produced by transfected cell lines. The carbohydrate attachment site of plasma apoE was localized to a single tryptic peptide (residues 192-206). Sequence analysis and amino sugar analysis of this peptide derived from asialo-, monosialo-, or disialo-apoE indicated that the carbohydrate moiety is attached only to Thr194 in monosialo- and disialo-apoE and that asialo-apoE is not glycosylated. Mammalian cells that normally do not express apoE were transfected with human apoE plasmid expression vectors to test the utilization of potential carbohydrate attachment sites and the role of apoE glycosylation in secretion. Site-specific mutants of apoE, designed to eliminate or alter glycosylation sites, were expressed in HeLa cells by acute transfection. Apolipoprotein E(Thr194----Ala) was secreted exclusively as the asialo isoform, confirming that Thr194 is the site of carbohydrate attachment in these cells and indicating that glycosylation of apoE is not essential for secretion. Apolipoprotein E(Thr194----Asn,Gly196----Ser), which introduces a potential site for N-glycosylation at position 194, was secreted with a higher apparent molecular weight than native, O-glycosylated apoE. Studies with tunicamycin indicated that this apoE was N-glycosylated at Asn194. Stably transfected cell lines expressing human apoE were prepared from wild-type Chinese hamster ovary (CHO) cells and from CHO ldlD cells, which are defective in glycosylation. The transfected wild-type cells secreted multiply sialylated apoE. The transfected ldlD cells also secreted high levels of apoE even in the absence of glycosylation, which confirms that glycosylation is not essential for secretion of apoE.     

Dissecting the domain structure of the regulatory subunit of cAMP-dependent protein kinase I and elucidating the role of MgATP

A truncated regulatory subunit of cAMP-dependent protein kinase I was constructed which contained deletions at both the carboxyl terminus and at the amino terminus. The entire carboxyl-terminal cAMP-binding domain was deleted as well as the first 92 residues up to the hinge region. This monomeric truncated protein still forms a complex with the catalytic subunit, and activation of this complex is mediated by cAMP. The affinity of this mutant holoenzyme for cAMP and its activation by cAMP are nearly identical to holoenzyme formed with a regulatory subunit having only the carboxyl-terminal deletion and very similar to native holoenzyme. The off rate for cAMP from both mutant regulatory subunits, however, is monophasic and very fast relative to the biphasic off rate seen for the native regulatory subunit. The effects of NaCl, urea, and pH on cAMP binding are also very similar for the mutant and native holoenzymes. Like the native type I holoenzyme, both mutant holoenzymes bind ATP with a high affinity. The positive cooperativity seen for MgATP binding to the native holoenzyme, however, is abolished in the double deletion mutant. The Hill coefficient for ATP binding to this mutant holoenzyme is 1.0 in contrast to 1.6 for the native holoenzyme. The Kd (cAMP) is increased by approximately 1 order of magnitude for both mutant forms of the holoenzyme in the presence of MgATP. A similar shift is seen for the native holoenzyme. Further characterization of the MgATP-binding properties of the wild-type holoenzyme indicates that a binary complex containing catalytic subunit and MgATP is required, in particular, for reassociation with the cAMP-bound regulatory subunit. This binary complex is required for rapid dissociation of the bound cAMP and is probably responsible for the observed reduction in cAMP-binding affinity for the type I holoenzyme in the presence of MgATP.     

Isolation and characterization of a glycosylated form of human insulin-like growth factor I produced in Saccharomyces cerevisiae

Expression and secretion of human insulin-like growth factor-I (IGF-I) in Saccharomyces cerevisiae was achieved by linking an actin (ACT) promoter to an MF alpha 1 prepro leader peptide/IGF-I gene fusion. Purified human IGF-I from yeast culture media was found to contain, in addition to the native form, also a glycosylated variant. Structural studies showed that both IGF-I forms were processed identically, resulting in 70-amino-acid long polypeptides, with intact N-terminal and C-terminal residues of glycine and alanine, respectively. The glycosylation site was determined to threonine-29 (Thr29), by 1H NMR spectroscopy and protein sequence analysis of an isolated tryptic peptide(22-36). No other glycosylation sites were found. Only mannose was detected in the sugar analysis, with an estimated content of 4.5% w/w corresponding to 2 mannose residues per molecule of IGF-I. The carbohydrate structure, determined by 1H and 13C NMR spectroscopy, was found to be alpha-D-Manp(1----2)alpha-D-Manp(1----3)Thr corresponding to an O-linked glycoprotein structure. No other post-translational modifications could be identified in the glycosylated IGF-I form. Furthermore, this form was highly active, comparable to native IGF-I, exhibiting a specific activity of 20,500 units/mg, as determined by a radio-receptor assay.     

The multiple membrane spanning topography of the beta 2-adrenergic receptor. Localization of the sites of binding, glycosylation, and regulatory phosphorylation by limited proteolysis

The beta 2-adrenergic receptor (beta-AR) is an integral membrane glycoprotein of apparent Mr approximately equal to 64,000. The amino acid sequence deduced from the beta-AR gene reveals homology with the visual pigment rhodopsin of retinal rod outer segments. We have proposed a structural model of beta-AR which is similar to that elucidated for rhodopsin. In this paper we identify a number of structural and topographical characteristics of beta-AR consistent with the model through the use of limited proteolysis. Limited trypsinization of beta-AR reconstituted in lipid vesicles yields two insoluble (integral membrane) domains of Mr approximately equal to 38,000 and 26,000. Identical results were obtained in intact cells, indicating that the cleavage site of the receptor is accessible at the extracellular surface of the plasma membrane. The amino-terminal domain (38 kDa) contains the ligand binding site (as revealed by photoaffinity labeling) and the sites of glycosylation (as revealed by its sensitivity to endoglycosidase F), whereas the carboxyl-terminal domain (26 kDa) contains all the sites of in vitro phosphorylation by cAMP-dependent protein kinase and the beta-adrenergic receptor kinase. Of four canonical sites for N-linked glycosylation, two near the amino and two near the carboxyl terminus, only those in the amino-terminal domain (Asn6 and Asn15) are utilized and sensitive to endoglycosidase F. Carboxypeptidase Y treatment of reconstituted native beta-adrenergic receptor generates a truncated (approximately 57 kDa) glycopeptide that has lost most of the sites phosphorylated by beta-AR kinase and one of the sites phosphorylated by protein kinase A. The various features delineated, including the length of the carboxypeptidase Y-sensitive region, the extracellular location of the trypsin-sensitive site, the location of the sites of phosphorylation and glycosylation all constrain the receptor to a rhodopsin-like structure with multiple membrane spanning segments.     

Identification and partial characterization of multiple native and neoantigenic epitopes of human C-reactive protein by using monoclonal antibodies

Multiple mAb to human C-reactive protein (CRP) were prepared which reacted preferentially with either native CRP, modified CRP (expressing "neo-CRP" determinants) or both forms of the molecule. These mAb were divided into four groups according to their binding characteristics to various CRP preparations and CRP peptides by using a combination of ELISA, dot blot, and Western blot assays; they were further characterized based upon their reactivity with CRP in the presence of calcium and inhibition by phosphorylcholine. The first group consisted of mAb that reacted only with native CRP, and served to define four distinct native CRP epitopes. The second group consisted of mAb that reacted with native CRP and also with CRP modified by direct immobilization on polystyrene plates, urea-chelation or SDS treatment in the absence of calcium, thus identifying a fifth native CRP epitope; these mAb displayed significantly greater reactivity with native than with modified CRP. The third group included mAb that reacted only with modified CRP and with the larger amino-terminal fragment (residues 1-146) of pronase-cleaved CRP. The fourth group included mAb that reacted only with modified CRP and with the smaller carboxyl-terminal fragment (residues 147-206) of pronase-cleaved CRP; most of these antibodies also reacted with the carboxyl-terminal octapeptide (residues 199-206) of CRP. These experiments have identified mAb that react preferentially with distinct conformational and sequence-determined epitopes of native and modified forms of the CRP molecule, respectively; provide partial identification of the epitopes with which they interact; point to the presence of at least five epitopes on native CRP and at least three epitopes on modified CRP; and provide antibodies suitable for identification and quantitation of native and modified forms of CRP. The mAb directed against neo-CRP epitopes may help identify the presence of this pentraxin and antigenically-related proteins at previously unappreciated sites.     

The role of the polar, carboxyl-terminal domain of Escherichia coli leader peptidase in its translocation across the plasma membrane

Leader peptidase, an integral membrane protein of Escherichia coli, is made without a cleavable leader sequence. It has 323 amino acid residues and spans the plasma membrane with a small amino-terminal domain exposed to the cytoplasm and a large, carboxyl-terminal domain exposed to the periplasm. We have investigated which regions of leader peptidase are necessary for its assembly across the membrane. Deletions were made in the carboxyl-terminal domain of leader peptidase, removing residues 141-222, 142-323, or 222-323. Protease accessibility was used to determine whether the polar, carboxyl-terminal domains of these truncated leader peptidases were translocated across the membrane. The removal of either residues 222-323 (the extreme carboxyl terminus) or residues 141-222 does not prevent leader peptidase membrane assembly. However, leader peptidase lacking both regions, i.e. amino acid residues 142-323, cannot translocate the remaining portion of its carboxyl terminus across the membrane. Our data suggest that the polar, periplasmic domain of leader peptidase contains information which is needed for membrane assembly.     

Alternatively spliced form of human thyroperoxidase, TPOzanelli: activity, intracellular trafficking, and role in hormonogenesis

Thyroperoxidase (TPO), a type I transmembrane heme containing glycoprotein, catalyzes iodide organification and thyroid hormone synthesis. One of the two main alternatively spliced forms of this enzyme, TPOzanelli, which is present in Graves's disease thyroid tissue, has a cytoplasmic domain completely modified. In the first stage of this study, the results of RT-PCR experiments showed that the TPOzanelli mRNA is present in normal thyroid tissue. We then generated CHO cell lines expressing the wild-type TPO (TPO1) and the alternatively spliced form TPOzanelli. Upon investigating a panel of 12 mAbs directed against the extracellular domain of TPO1 and sera from patients with a high titer of TPO autoantibodies, we observed that (i) the three-dimensional structure of this domain is similar in both isoforms; (ii) the autoantibodies recognize TPOzanelli as well as TPO1. The results of pulse chase and cell surface biotinylation experiments showed that the TPOzanelli has a shorter half-life (7 versus 11 h) and is expressed at the cell surface in lesser amounts than TPO1 (7 versus 15%). The total enzymatic activity and cell surface activity were determined in CHO cells expressing TPO1 and TPOzanelli, and TPO1 and TPOzanelli were found to have similar levels of activity. It was established that approximately 20% of the TPO purified from a Graves' disease thyroid gland was precipitated by polyclonal antibodies directed against a specific part of the cytoplasmic tail of TPOzanelli. This confirmed that the protein corresponding to the mRNA is present in the thyroid tissue. All in all, these results indicate that TPOzanelli can be expected to play a role in thyroid hormone synthesis and in thyroid autoimmunity.     

The proximal carboxyl-terminal domains of ADAMTS13 determine substrate specificity and are all required for cleavage of von Willebrand factor

ADAMTS13 limits platelet-rich thrombosis by cleaving von Willebrand factor at the Tyr(1605)-Met(1606) bond. Previous studies showed that ADAMTS13 truncated after spacer domain remains proteolytically active or hyperactive. However, the relative contribution of each domain within the proximal carboxyl terminus of ADAMTS13 in substrate recognition and specificity is not known. We showed that a metalloprotease domain alone was unable to cleave the Tyr-Met bond of glutathione S-transferase (GST)-VWF73-H substrate in 3 h, but it did cleave the substrate at a site other than the Tyr-Met bond after 16-24 h of incubation. Remarkably, the addition of even one or several proximal carboxyl-terminal domains of ADAMTS13 restored substrate specificity. Full proteolytic activity, however, was not achieved until all of the proximal carboxyl-terminal domains were added. The addition of TSP1 2-8 repeats and two CUB domains did not further increase proteolytic activity. Furthermore, ADAMTS13 truncated after the spacer domain with or without metalloprotease domain bound GST-VWF73-H with a K(d) of approximately 7.0 or 13 nm, comparable with full-length ADAMTS13 (K(d) = 4.6 nm). Metalloprotease domain did not bind GST-VWF73-H detectably, but the disintegrin domain, first TSP1 repeat, Cys-rich domain, and spacer domain bound GST-VWF73-H with K(d) values of 489, 136, 121, and 108 nm, respectively. These proximal carboxyl-terminal domains dose-dependently inhibited cleavage of fluorescent resonance energy transfer (FRETS)-VWF73 by full-length ADAMTS13 and ADAMTS13 truncated after the spacer domain. These data demonstrated that the proximal carboxyl-terminal domains of ADAMTS13 determine substrate specificity and are all required for recognition and cleavage of von Willebrand factor between amino acid residues Asp(1595) and Arg(1668).     

Secretion of heparanase protein is regulated by glycosylation in human tumor cell lines

The endo-beta-d-glucuronidase, heparanase, is capable of specifically degrading heparan sulfate, and this activity is associated with the metastatic potential of tumor cells. The predicted amino acid sequence of heparanase includes six putative N-glycosylation sites; however, the precise biochemical role of glycosylated heparanase remains unknown. In this study, we examined the link between glycosylation and the function of heparanase in human tumor cell lines. Heparanase protein was glycosylated at six Asn residues in human tumor cell lines. Treatment with a glycosylation inhibitor demonstrated that glycosylation was not required for the activity of heparanase. However, glycosylation affected the kinetics of endoplasmic reticulum-to-Golgi transport and of secretion of the enzyme.