The effect of culture medium and carrier on explant culture of human limbal epithelium: A comparison of ultrastructure, keratin profile and gene expression.

Patients with limbal stem cell deficiency (LSCD) often experience pain and photophobia due to recurrent epithelial defects and chronic inflammation of the cornea. Successfully restoring a healthy corneal surface in these patients by transplantation of ex vivo expanded human limbal epithelial cells (LECs) may alleviate these symptoms and significantly improve their quality of life. The clinical outcome of transplantation is known to be influenced by the quality of transplanted cells. Presently, several different protocols for cultivation and transplantation of LECs are in use. However, no consensus on an optimal protocol exists. The aim of this study was to examine the effect of culture medium and carrier on the morphology, staining of selected keratins and global gene expression in ex vivo cultured LECs. Limbal biopsies from cadaveric donors were cultured for three weeks on human amniotic membrane (HAM) or on tissue culture coated plastic (PL) in either a complex medium (COM), containing recombinant growth factors, hormones, cholera toxin and fetal bovine serum, or in medium supplemented only with human serum (HS). The expanded LECs were examined by light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) for keratins K3, K7, K8, K12, K13, K14, K15 and K19, as well as microarray and qRT-PCR analysis. The cultured LECs exhibited similar morphology and keratin staining on LM, TEM and IHC examination, regardless of the culture condition. The epithelium was multilayered, with cuboidal basal cells and flattened superficial cells. Cells were attached to each other by desmosomes. Adhesion complexes were observed between basal cells and the underlying carrier in LECs cultured on HAM, but not in LECs cultured on PL. GeneChip Human Gene 2.0 ST microarray (Affymetrix) analysis revealed that 18,653 transcripts were ≥2 fold up or downregulated (p ≤ 0.05). Cells cultured in the same medium (COM or HS) showed more similarities in gene expression than cells cultured on the same carrier (HAM or PL). When each condition was compared to HAM/COM, no statistical difference was found in the transcription level of the selected genes associated with keratin expression, stemness, proliferation, differentiation, apoptosis, corneal wound healing or autophagy. In conclusion, the results indicate that ex vivo cultures of LECs on HAM and PL, using culture media supplemented with COM or HS, yield tissues with similar morphology and keratin staining. The gene expression appears to be more similar in cells cultured in the same medium (COM or HS) compared to cells cultured on the same carrier (HAM or PL).

Oxidized LDL level is related to gene expression of tumour necrosis factor super family members in children and young adults with familial hypercholesterolaemia.

OBJECTIVE: Familial hypercholesterolaemia (FH) is associated with increased risk of premature atherosclerosis. Inflammation is a key event in atherogenesis, and we have previously reported an inflammatory imbalance between tumour necrosis factor (TNF)α and interleukin-10 in children with FH. Based on the potential role of TNF-related molecules in inflammation, we investigated the regulation of other members of the TNF superfamily (TNFSF)/TNF receptor superfamily (TNFRSF) in children and young adults with FH and matched healthy controls. METHODS: Expression of TNFSF/TNFRSF genes in peripheral blood mononuclear cells (PBMCs) was quantified in children and young adults with FH prior to (n = 42) and after statin treatment (n = 10) and in controls (n = 25) by quantitative real-time polymerase chain reaction. RESULTS: First we found that, compared with controls, the mRNA levels of OX40L, BAFFR and TRAILR1 were significantly higher, whereas TRAIL and TRAILR3 were significantly lower in children and young adults with FH. Secondly, levels of oxidized low-density lipoprotein (oxLDL) were significantly raised in the FH group, and correlated with the expression of OX40L, BAFFR and TRAILR1. Thirdly, oxLDL increased mRNA levels of BAFFR, TRAILR1 and TRAILR4 in PBMCs ex vivo from individuals with FH. Fourthly, OX40, acting through OX40L, enhanced the oxLDL-induced expression of matrix metalloproteinase-9 in THP-1 monocytes in vitro. Finally, after statin treatment in children with FH (n = 10), mRNA levels of OX40L and TRAILR1 decreased, whereas levels BAFF, TRAIL and TRAILR3 increased. CONCLUSION: Our findings suggest the involvement of some TNFSF/TNFRSF members and oxLDL in the early stages of atherogenesis; this may potentially contribute to the accelerated rate of atherosclerosis observed in individuals with FH.

Gene expression and distribution of key bone turnover markers in the callus of estrogen-deficient, vitamin D-depleted rats.

An experimental rat model was used to test the hypothesis that in osteoporosis (OP) the molecular composition of the extracellular matrix in the fracture callus is disturbed. OP was induced at 10 weeks of age by ovariectomy and a vitamin D(3)-deficient diet, and sham-operated animals fed normal diet served as controls. Three months later a closed tibial fracture was made and stabilized with an intramedullary nail. After 3 and 6 weeks of healing, the animals were killed and the fracture calluses examined with global gene expression, in situ mRNA expression, and ultrastructural protein distribution of four bone turnover markers: osteopontin, bone sialoprotein, tartrate-resistant acid phosphatase, and cathepsin K. Global gene expression showed a relatively small number of differently regulated genes, mostly upregulated and at 3 weeks. The four chosen markers were not differently regulated, and only minor differences in the in situ mRNA expression and ultrastructural protein distribution were detected. Gene expression and composition of fracture calluses are not generally disturbed in experimental OP.

The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway.

Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin's potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated.

Binding and cyclic AMP stimulation by N-terminally deleted human PTHs (3-84 and 4-84) in a homologous ligand receptor system.

We have produced in yeast two human parathyroid hormone (hPTH) analogs with amino-terminal deletions, hPTH(3-84) and hPTH(4-84), employing the mating factor alpha (MF alpha) expression system. The authenticity of the polypeptides was demonstrated by amino-terminal analysis, amino acid composition, and molecular mass analysis. In cells (LLC-PK1) transfected with the human PTH/parathyroid hormone-related protein (PTHrP) receptor, using [125I-Tyr36]chickenPTHrP(1-36)NH2 as radioligand, binding studies revealed dissociation constants at equilibrium (Kd) for hPTH(3-84) and hPTH(4-84) of 4.7 and 8.0 nM, respectively, only slightly higher than natural recombinant hPTH(1-84) Kd = 2.3 nM). In comparison, [Nle8,18,Tyr34]bovinePTH(3-34)NH2 and [Tyr36]cPTHrP(1-36)NH2 showed equal Kd's of 1.9 nM. Neither of the N-terminally deleted hPTH analogs showed any detectable stimulation of cAMP production in the cells at concentrations below 20 nM. At supersaturated concentrations (500 nM) with receptor occupancy of more than 95% these hPTH analogs revealed about 15% rest agonism compared with that of hPTH(1-84). hPTH(1-84) and [Tyr36]cPTHrP(1-36)NH2 showed an equal half maximal cyclic adenosine monophosphate (cAMP) stimulation of about 0.8 and 0.7 nM, respectively. The hPTH analogs did not show any ability to antagonize cellular cAMP production induced by either hPTH or [Tyr36]cPTHrP(1-36)NH2. [Nle8,18,Tyr34]bPTH(3-34)NH2 did also not antagonize cAMP stimulation by hPTH, but inhibited [Tyr36]cPTHrP(1-36)NH2-induced cAMP production by 40% when present at a 1000 M excess. These distinct results related to PTH and PTHrP from different species are important to consider in experiments evaluating potential hPTH or PTHrP antagonism, and employment of a hPTH/PTHrP receptor model is a requirement.

Sox-4 messenger RNA is expressed in the embryonic growth plate and regulated via the parathyroid hormone/parathyroid hormone-related protein receptor in osteoblast-like cells.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) exert potent and diverse effects in cells of the osteoblastic and chondrocytic lineages. However, downstream mediators of these effects are characterized inadequately. We identified a complementary DNA (cDNA) clone encoding the 5' end of the transcription factor Sox-4, using a subtracted cDNA library enriched in PTH-stimulated genes from the human osteoblast-like cell line OHS. The SOX-4 gene is a member of a gene family (SOX and SRY) comprising transcription factors that bind to DNA through their high mobility group (HMG)-type binding domain, and previous reports have implicated Sox proteins in various developmental processes. In situ hybridization of fetal and neonatal mouse hindlimbs showed that Sox-4 messenger RNA (mRNA) was expressed most intensely in the zone of mineralizing cartilage where chondrocytes undergo hypertrophy, and by embryonic day 17 (ED17), after the primary ossification center was formed, its expression was detected only in the region of hypertrophic chondrocytes. Sox-4 mRNA was detected in osteoblast-like cells of both human and rodent origin. In OHS cells, physiological concentrations (10(-10)-10(-9) M) of human PTH 1-84 [hPTH(1-84)] and hPT(1-34), but not hPTH(3-84), stimulated Sox-4 mRNA expression in a time-dependent manner, indicating involvement of the PTH/PTHrP receptor. Sox-4 transcripts also were detected in various nonosteoblastic human cell lines and tissues, in a pattern similar to that previously reported in mice. The presence of Sox-4 mRNA in hypertrophic chondrocytes within the mouse epiphyseal growth plate at sites that overlap or are adjacent to target cells for PTH and PTHrP, and its strong up-regulation via activated PTH/PTHrP receptors in OHS cells, makes it a promising candidate for mediating downstream effects of PTH and PTHrP in bone.

Two human osteoblast-like osteosarcoma cell lines show distinct expression and differential regulation of parathyroid hormone-related protein.

Parathyroid hormone (PTH)-related protein (PTHrP) acts as a local regulator of osteoblast function via mechanisms that involve PTH/PTHrP receptors linked to protein kinase A (PKA) and C (PKC). However, the regulation of PTHrP production and mRNA expression in human osteoblasts is poorly understood. Here we have characterized alternative PTHrP mRNA 3' splicing variants, encoding PTHrP isoforms of 139, 141, and 173 amino acids, and studied the regulation of PTHrP and its mRNAs by activated PKA and PKC in two human osteoblast-like cell lines (KPDXM and TPXM). Using exon-specific Northern analysis and reverse transcriptase-coupled polymerase chain reaction, we identified mRNAs encoding PTHrP(1-139) and PTHrP(1-141) in both cell lines. PTHrP(1-139) mRNAs predominated in TPXM cells and PTHrP(1-173) mRNAs were only detected in TPXM cells. Activation of PKA or PKC resulted in different effects on PTHrP and its mRNAs in the two cell lines. In TPXM cells, peptide-specific immunoassays detected high basal levels of PTHrP, increasing by 2-fold in cell extracts and 4-fold in culture media at 7 h and 24 h after exposure to forskolin, respectively, paralleling changes in PTHrP mRNA expression. Phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), a PKC activator, had no effect. In KPDXM cells, PTHrP was not detected in culture media under basal experimental conditions, and barely detectable amounts were present in cell extracts of TPA-treated cells, although the mRNA levels increased substantially in response to TPA. In the responsive cell lines, the effects on mRNA levels were dose dependent, and increased by 6.9- to 10.5-fold and 2.0- to 4.1-fold at 4 h in TPXM and KPDXM cells after exposure to 10 microM forskolin and 150 nM TPA, respectively. PTHrP mRNA levels then declined but were sustained above controls also at 12 h in both cell lines, albeit at considerably higher levels in TPXM cells. The different responsiveness to agents activating PKA- and PKC-dependent pathways may depend on the cellular state of differentiation, or alternatively, cancer cell line-specific defects. Our data demonstrating distinct differences in mRNA species and the amounts of PTHrP produced by the two cell lines as compared with roughly equivalent overall mRNA levels may suggest that post-transcriptional mechanisms play an important role in limiting the production of intracellular and secreted PTHrPs in human osteoblastic cells.

High-level production of human parathyroid hormone in Bombyx mori larvae and BmN cells using recombinant baculovirus.

A full-length cDNA encoding human parathyroid hormone (hPTH) containing the prepro region was cloned into Bombyx mori baculovirus under the control of the polyhedrin promoter and polyadenylation sequences. After transfection and generation of the recombinant baculovirus, hPTH production was examined in silkworm larvae and BmN cell cultures. The larvae synthesized and efficiently secreted the correctly processed and authentic hPTH (9.4 kDa) with no sign of internal degradation. In BmN cells, the major secreted form was the correctly sized protein, but small amounts of degraded hPTH could also be detected in the medium by immunoblotting. Unlike the situation in larvae, prepro-hPTH could also be demonstrated intracellularly in BmN cells. The concentration of hPTH in the larval hemolymph was about 70 mg/l, as compared to approx. 55 micrograms/l in the medium per 7.5 x 10(6) cells. Recombinant hPTH (re-hPTH) from the hemolymph was purified by reverse-phase HPLC and subjected to chemical and biological analyses. The authenticity of the purified re-hPTH was confirmed by N-terminal sequencing, amino acid composition and a mass of 9425 Da, close to the theoretical value. The hormone showed high-affinity receptor binding and full biological potency in increasing cellular cAMP.

Expression and characterization of a recombinant human parathyroid hormone partial agonist with antagonistic properties: Gly-hPTH(-1-->+84).

We have produced and characterized a hPTH analogue with an amino-terminal extension of glycine, Gly-hPTH(-1-->+84) (denoted Gly-hPTH). The hormone analogue was synthesized in E. coli strain BJ5183 transformed with the expression plasmid pKKPTH, extracted from the bacterial pellet and purified by reverse-phase high performance liquid chromatography. Its chemical nature, as determined by amino acid composition analysis, N-terminal amino acid analysis, and mass spectrometry, showed the 9480-Da Gly-hPTH as the predominant species. Because f-Met-Gly-hPTH was the expected form encoded by the plasmid construct, the results indicate that the f-Met residue was efficiently removed from the precurser form. The following functional characteristics of Gly-hPTH were demonstrated. 1) In cells transfected with the human PTH/PTHrP receptor, the receptor binding affinity was reduced threefold compared to the authentic hPTH(1-84) produced by Saccharomyces cerevisiae (apparent Kds: 8.4 and 2.7 nM, respectively). 2) Using the same cells, Gly-hPTH showed 27-fold reduced potency compared to hPTH(1-84) in stimulating intracellular cAMP production (EC50: 32 and 1.2 nM, respectively). 3) Gly-hPTH demonstrated antagonist activity by reducing hPTH-induced cAMP production by 33 +/- 5% (mean +/- SD) when tested at a 1:1 molar ratio. In these studies the recombinant authentic hPTH(1-84) was used as standard for comparisons, and it showed an equal receptor binding affinity and cAMP production as the chemically synthesized peptide [Nle8,18,Tyr34]bovinePTH(1-34)-NH2.

Differences in binding affinities of human PTH(1-84) do not alter biological potency: a comparison between chemically synthesized hormone, natural and mutant forms.

The purpose of this study was to evaluate receptor binding affinities and biological properties in vitro and in vivo of various recombinant hPTH(1-84) forms representing the natural hormone and a mutagenized hPTH form, [Gln26]hPTH(1-84) (QPTH), after expression in E. coli and Saccharomyces cerevisiae. In LLC-PK1 cells stably transformed with the rat PTH/PTHrP receptor, chemically synthesized hPTH(1-84) and QPTH showed a reduced binding affinity (apparent Kd 18 and 23 nM, respectively) than the recombinant, hPTH(1-84) (apparent Kd 9.5 nM). All recombinant hPTH forms showed a similar potency to stimulate cellular cAMP production (EC50 1.5 nM) and significantly better than chemically synthesized hPTH (EC50 5.7 nM). All hormone forms showed an about equipotent activity in causing elevation in serum calcium, increased excretion of urine phosphate, and cAMP. Thus, the natural recombinant PTH forms showed higher binding affinities and adenylate cyclase activation potencies in LLC-PK1 cells, but the reduced receptor binding affinity exerted by QPTH did not transcend differences in cAMP generation and in vivo biological activities.

Expression of human parathyroid hormone in mammalian cells, Escherichia coli and Saccharomyces cerevisiae.

The entire human parathyroid hormone (hPTH) cDNA gene with its natural signal and pro-region is expressed in transfected mouse mammary tumor cells (C127I cells) and Chinese hamster lung cells (DON cells) under control of the murine metallothioneine-1 promoter in a vector in which replication functions are provided by the entire genome of bovine papilloma virus type I (BPV-1). Authentic hPTH is efficiently produced by the non-endocrine cells and secreted to the growth medium without any abberant processing. Immunoblots from SDS-PAGE gels of concentrated growth medium reveal one band corresponding to intact, undegraded hPTH. Purification by reversed-phase HPLC results in a peptide with an amino acid content and N-terminal sequence identical to hPTH. For comparison, hPTH cDNA with deleted prepro-region is also expressed as secretory proteins in Escherichia coli and in Saccharomyces cerevisiae. In E. coli the vector construct is based on the staphylococcal protein A promoter employing protein A signal sequence. In S. cerevisiae a mating factor alpha expression system containing the factor alpha-signal sequence is employed. The results show that intact hPTH is secreted in addition to proteolytically cleaved fragments in both microorganisms. Thus, the signal sequences promote efficient secretion, and correct N-terminal processing of hPTH in both mammalian, bacterial and yeast cells. However, the folding characteristics of hPTH make it susceptible to internal proteolytical cleavage which appears to be species specific in yeast and E. coli.

Amino acid sequences in amyloid proteins of kappa III immunoglobulin light-chain origin.

The main amyloid fibril (AL) proteins extracted from the spleen of Patient So 124 with systemic amyloidosis and from a skin nodule of Patient KSA with localized amyloidosis were studied by partial amino acid sequence analysis and proved to be of kappa III immunoglobulin light-chain origin. The sequences were similar to that of Bence Jones protein V and, which has been reported to have a unique kappa III subset sequence. Thus, except for position 9 in protein AL(KSA), the amino acid sequences were identical to position 25 in AL(So 124) and in AL(KSA). The question is being raised whether this kappa III subset might contain amyloidogenic sequences.

Protein kinase C and cyclic AMP-dependent protein kinase phosphorylate phospholemman, an insulin and adrenaline-regulated membrane phosphoprotein, at specific sites in the carboxy terminal domain.

Phospholemman, a transmembrane, 72 residue protein enriched in striated muscle and heart [Palmer, Scott and Jones (1991) J. Biol. Chem. 266, 11126-11130], is phosphorylated in response to insulin [Walaas, Horn and Walaas (1991) Biochim. Biophys. Acta 1094, 92-102]. The present study is aimed at identifying the phosphorylation sites of this protein. A synthetic peptide, GTFRSS63IRRLS68TRRR (in the single letter code) and consisting of phospholemman residues 58-72, is a substrate for both protein kinase C and cyclic AMP (cAMP)-dependent protein kinase, with Km values of 6-7 microM for both enzymes. Amino acid sequencing of the phosphopeptide shows that protein kinase C phosphorylates both Ser-63 and Ser-68, while cAMP-dependent protein kinase phosphorylates Ser-68. Thermolytic phosphopeptide mapping of 32P-labelled phospholemman from rat diaphragms shows that treatment with insulin results in labelling of phosphopeptides containing both Ser-63 and Ser-68, whereas treatment with adrenaline results in labelling of the phosphopeptide containing Ser-68. Hence, insulin and adrenaline regulate the phosphorylation of phospholemman, presumably through protein kinase C and cAMP-dependent protein kinase, respectively, on partly overlapping phosphorylation sites.

Synthesis of human parathyroid-hormone-related protein(1-141) in Saccharomyces cerevisiae. A correct amino-terminal processing vital for the hormone's biological activity is obtained by an ubiquitin fusion protein approach.

Gene fusions have been widely used in heterologous expression systems as a technique to stabilize the recombinant product against proteolysis, increase the translational initiation efficiency or to serve as an affinity handle for the purification of the protein. A further advantage is the potential to generate an authentic amino terminus of the foreign protein when this is vital for its biological activity, such as for the ability of human parathyroid-hormone-related protein (hPTHrP) to mediate activation of adenylate cyclase. We report here the construction and utility of a ubiquitin fusion protein system for production of the otherwise short-lived hPTHrP(1-141) as a carboxyl extension to ubiquitin in yeast. A hybrid gene containing the hPTHrP(1-141) cDNA coding region fused in-frame to the 3' end of the yeast ubiquitin cDNA was constructed and expressed under the control of the regulatable yeast metallothionein promoter. The recombinant protein was purified to homogeneity and finally characterized by N-terminal amino acid sequencing and amino acid composition analysis, demonstrating that the fusion protein was cleaved correctly and quantitatively in vivo by an ubiquitin-specific yeast endoprotease to generate authentic hPTHrP(1-141). hPTHrP(1-141) stimulated adenylate cyclase in rat osteosarcoma cell membranes to the same extent as equimolar amounts of recombinant human parathyroid hormone(1-84) and [Tyr34]hPTHrP(1-34)amide. Thus, this expression cloning strategy permits the production of authentic, biologically active recombinant hPTHrP(1-141), and the procedure can easily be adapted to make PTHrP analogues for further studies of its domain-specific activities and biological roles.

Parathyroid hormone-related protein is produced by cultured endothelial cells: a possible role in angiogenesis.

Parathyroid hormone-related protein (PTHrP) is produced by various normal and neoplastic tissues. Even if the physiological function(s) of PTHrP is unclear, evidence suggests that the protein may participate in the local regulation of smooth muscle contractility. We show here that PTHrP is produced in endothelial cells cultured from human umbilical veins as demonstrated both at the mRNA and protein level. The expression of PTHrP can be upregulated by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate, which is known to stimulate endothelial cell differentiation and angiogenesis in vitro. Unlike smooth muscle cells, the endothelial cells do not express the parathyroid hormone (PTH)/PTHrP receptor mRNA, nor could specific binding of the protein be detected. We therefore suggest that PTHrP produced by endothelial cells acts on smooth muscle cells and may be of importance for the growth and development of new vasculature.

Human parathyroid hormone as a secretory peptide in milk of transgenic mice.

In a transgenic mouse model we have targeted the expression of recombinant human parathyroid hormone (hPTH) to the mammary gland yielding hPTH as a secretory, soluble peptide in milk. A 2.5 kb upstream regulatory sequence of the murine whey acidic protein (WAP) directed the expression of the hPTH cDNA in a fusion gene construct (WAPPTHSV2) containing the SV40 small t-antigen intron and polyadenylation site in the 3' end. Established lines of transgenic mice secreted hPTH to milk in concentrations up to 415 ng/ml. Recombinant hPTH recovered from the milk was purified by HPLC and shown to be identical to hPTH standard as analyzed by SDS-PAGE followed by immunoblotting. Expression of the WAPPTHSV2 was limited to the mammary gland as analyzed by polymerase chain reaction (PCR) and Southern blot of reversed transcribed mRNA from different tissues. hPTH is an important bone anabolic hormone and may be a potentially important pharmaceutical for treatment of demineralization disorders such as osteoporosis. We present the transgenic animal as a possible production system for hPTH.

Characterization of a K26Q site-directed mutant of human parathyroid hormone expressed in yeast.

Human parathyroid hormone (hPTH) is susceptible to proteolytical cleavage both in humans and when expressed as a secretory product in Escherichia coli (Høgseth, A., Blingsmo, O. R., Saether, O., Gautvik, V. T., Holmgren, E., Hartmanis, M., Josephson, S., Gabrielsen, O. S., Gordeladze, J. O., Alestrøm, P., and Gautvik, K. M. (1990) J. Biol. Chem. 265, 7338-7344) and Saccharomyces cerevisiae (Gabrielsen, O. S., Reppe, S., Saether, O., Blingsmo, O. R., Sletten, K., Gordeladze, J. O., Høgset, A., Gautvik, V. T., Alestrøm, P., Oyen, T. B., and Gautvik, K. M. (1990) Gene (Amst.) 90, 255-262). In the latter system, one major site of cleavage was identified (Arg25-Lys26 decreased Lys27). To produce hPTH resistant to this proteolytic processing, a point mutation changing Lys26 to Gln was introduced, and the modified gene expressed in S. cerevisiae as a fusion protein with the alpha-factor leader sequence. The resulting major form of hPTH secreted to the growth medium was of full length showing that the mutation had eliminated internal processing. Consequently, the yield of the mutant hormone was significantly higher than obtained with the natural peptide. Using improved purification procedures, a significantly higher purity was also obtained. The secreted mutant hPTH-(1-84,Q26) had the correct size, full immunological reactivity with two different hPTH antisera, correct amino acid composition and N-terminal sequence, and correct mass as determined by mass spectrometry. Furthermore, the introduced mutation did not reduce the biological activity of the hormone as judged from its action in three biological assay systems: 1) a hormone-sensitive osteoblast adenylate cyclase assay; 2) an in vivo calcium mobilizing assay in rats; and 3) an in vitro bone resorption assay.

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.