Search

Heparan sulfate (HS) is an essential glycosaminoglycan (GAG) as a component of proteoglycans, which are present on the cell surface and in the extracellular matrix. HS-containing proteoglycans not only function as structural constituents of the basal lamina but also play versatile roles in various physiological processes, including cell signaling and organ development. Thus, inherited mutations of genes associated with the biosynthesis or degradation of HS can cause various diseases, particularly those involving the bones and central nervous system (CNS). Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders involving GAG accumulation throughout the body caused by a deficiency of GAG-degrading enzymes. GAGs are stored differently in different types of MPSs. Particularly, HS deposition is observed in patients with MPS types I, II, III, and VII, all which involve progressive neuropathy with multiple CNS system symptoms. While therapies are available for certain symptoms in some types of MPSs, significant unmet medical needs remain, such as neurocognitive impairment. This review presents recent knowledge on the pathophysiological roles of HS focusing on the pathogenesis of MPSs. We also discuss the possible use and significance of HS as a biomarker for disease severity and therapeutic response in MPSs.

Pabinafusp alfa (JR-141) is a novel enzyme drug that crosses the blood-brain barrier by transcytosis via transferrin receptors. In order to establish its efficacy and safety, a multicenter, single-arm, open-label phase 2/3 clinical trial was conducted in 28 Japanese patients with mucopolysaccharidosis II (MPS-II, Hunter syndrome) by intravenous administrations of 2.0 mg/kg of pabinafusp alfa for 52 weeks. The primary efficacy endpoint was changes in heparan sulfate (HS) concentrations in the cerebrospinal fluid (CSF). Secondary endpoints included assessments of neurocognitive development for central efficacy, and changes in plasma HS and dermatan sulfate (DS) concentrations for peripheral efficacy. HS concentrations in the CSF significantly decreased from baseline to week 52 (p < 0.001), suggesting continuous inhibition of substrate accumulations in the CNS, i.e., hitherto unaddressed progressive neurodegeneration. Evaluations of neurocognitive developments showed positive changes in 21 of the 28 patients. Serum HS and DS concentrations, liver and spleen volumes, and other assessments suggested the peripheral efficacy of pabinafusp alfa was comparable to that of idursulfase. Drug-related adverse events were mild or moderate in severity, transient, and manageable. The results establish delivery across the BBB of pabinafusp alfa as an effective therapeutic for treating both the CNS and peripheral symptoms of patients with MPS-II., Graphical Abstract, Neurodegeneration in mucopolysaccharidosis has not been amenable to the conventional enzyme replacement therapy due to the blood-brain barrier blocking the drug delivery into the brain. Pabinafusp alfa has been shown in this article to overcome this challenge with positive clinical neuropsychiatric outcomes in addition to its somatic efficacy.

Congenital scoliosis is defined by the presence of structural anatomical malformations that arise from failures of vertebral formation or segmentation before and after birth. The understanding of genetic background and key genes for congenital scoliosis is still poor. We herein report that the excess expression of plasminogen activator inhibitor-1 (Pai-1) induced by the upregulation of miR-224-5p is involved in the pathogenesis of congenital kyphoscoliosis through impaired osteoblast differentiation. We first investigated the variety and progression of abnormalities of the lumbar spines in Ishibashi (IS) rats, a rat model of congenital kyphoscoliosis. The rats had already shown fusion and division of the primary ossification center at postnatal day 4. Over time, the rats showed various abnormalities of the lumbar spine, including the fusion of the annular epiphyseal nucleus. At postnatal day 42, spinal curvature was clearly observed due to the fusion of the vertebral bodies. Using a microRNA array, we found that the expression of miR-224-5p was increased in the lumbar spine of the rats at postnatal day 4. The expression of Pai-1, which is involved in osteoblast differentiation regulated by miR-224-5p, was also increased, while the levels of type I collagen, a marker of osteoblast differentiation, were decreased in the lumbar spine. These results indicate that the aberrant expression of miRNA-224-5p and its target genes is involved in the impaired osteoblast differentiation and may provide a partial molecular explanation for the pathogenesis of congenital scoliosis.

Under clinical development for patients with growth hormone deficiency, JR-142 is a long-acting growth hormone with a half-life extended by fusion with modified serum albumin. We conducted a Phase 1 study to investigate the safety, tolerability, and pharmacokinetic (PK) and pharmacodynamic (PD) profiles of once-weekly subcutaneous administrations of JR-142. The study consisted of two parts: an open-label single ascending dosing study (Part 1), and a randomized, placebo-controlled, assessor-blinded multiple ascending dosing study (Part 2).A total of 31 healthy Japanese male participants were enrolled. In Part 1, seven of them received a single subcutaneous injection of JR-142 each at dosages of 0.15 mg/kg (n = 1), 0.25 mg/kg (n = 2), 0.5 mg/kg (n = 2), or 1.0 mg/kg (n = 2). In Part 2, one weekly subcutaneous injection of JR-142 at 0.25 mg/kg, 0.5 mg/kg, 1.0 mg/kg or a placebo were given for four weeks to each of the other 24 participants (six in each group). Plasma JR-142 and serum insulin-like growth factor-1 (IGF-1) concentrations were measured for PK and PD assessments. Safety was evaluated on the basis of adverse events (AEs), laboratory tests, and other measures.JR-142 induced dose-dependent increases in the maximum plasma JR-142 concentration (Csubmax/sub) and the area under the plasma concentration-time curve from time 0 to τ (AUCsub0-τ/sub). A similar dose-response relationship was observed in serum IGF-1 concentrations. All trough IGF-1 levels were well sustained one week after the final administrations of JR-142 at the three dosages, while the peak concentrations of IGF-1 remained mildly elevated. No serious AEs were observed, and laboratory tests, including assessment of anti-drug antibodies, uncovered no significant safety issues.Once-weekly subcutaneous injections of JR-142 produced positive dose-dependent PK and PD profiles over the dosage range. Drug accumulation was observed after the four-week administration period but did not raise safety concerns, indicating that JR-142 is well-tolerated in healthy participants. The PD profiles observed in terms of IGF-1 concentrations were also positive, and we believe the encouraging results of this study warrant substantiation in further clinical trials in patients with GHD.This clinical study was conducted at one investigational site in Osaka, Japan, where the clinical study and the non-clinical data of JR-142 were reviewed and approved by its Institutional Review Board on 9th May 2019. The study was conducted in compliance with the approved study protocol, the Declaration of Helsinki, 1964, as revised in 2013, and Good Clinical Practice.

Whereas significant strides have been made in the treatment of lysosomal storage diseases (LSDs), the neuronopathy associated with these diseases remains impervious mainly because of the blood-brain barrier (BBB), which prevents delivery of large molecules to the brain. However, 100 years of research on the BBB since its conceptualization have clarified many of its functional and structural characteristics, spurring recent endeavors to deliver therapeutics across it to treat central nervous system (CNS) disorders, including neuronopathic LSDs. Along with the BBB, the cerebrospinal fluid (CSF) also functions to protect the microenvironment of the CNS, and it is therefore deeply involved in CNS disorders at large. Recent research aimed at developing therapeutics for neuronopathic LSDs has uncovered a number of critical roles played by the CSF that require further clarification. This review summarizes the most up-to-date understanding of the BBB and the CSF acquired during the development of therapeutics for neuronopathic LSDs, and highlights some of the associated challenges that require further research.

Enzyme replacement therapy (ERT) improves somatic manifestations in mucopolysaccharidoses (MPS). However, because intravenously administered enzymes cannot cross the blood–brain barrier (BBB), ERT is ineffective against the progressive neurodegeneration and resultant severe central nervous system (CNS) symptoms observed in patients with neuronopathic MPS. Attempts to surmount this problem have been made with intrathecal and intracerebroventricular ERT in order to achieve CNS effects, but the burdens on patients are inimical to long-term administrations. However, since pabinafusp alfa, a human iduronate-2-sulfatase fused with a BBB-crossing anti-transferrin receptor antibody, showed both central and peripheral efficacy in a mouse model, subsequent clinical trials in a total of 62 patients with MPS-II (Hunter syndrome) in Japan and Brazil substantiated this dual efficacy and provided an acceptable safety profile. To date, pabinafusp alfa is the only approved intravenous ERT that is effective against both the somatic and CNS symptoms of patients with MPS-II. This article summarizes the previously obtained preclinical and clinical evidence related to the use of this drug, presents latest data, and discusses the preclinical, translational, and clinical challenges of evaluating, ameliorating, and preventing neurodegeneration in patients with MPS-II.

In Hunter syndrome (mucopolysaccharidosis II [MPS-II]), systemic accumulation of glycosaminoglycans (GAGs) due to a deficiency of iduronate-2-sulfatase (IDS), caused by mutations in the IDS gene, leads to multiple somatic manifestations and in patients with the severe (neuronopathic) phenotype, also to central nervous system (CNS) involvement. These symptoms cannot be effectively treated with current enzyme-replacement therapies, as they are unable to cross the blood-brain barrier (BBB). Pabinafusp alfa, a novel IDS fused with an anti-human transferrin receptor antibody, was shown to penetrate the BBB and to address neurodegeneration in preclinical studies. Subsequent phase 1/2 and 2/3 clinical studies in Japan have shown marked reduction of GAG accumulation in the cerebrospinal fluid (CSF), along with favorable clinical responses. A 26-week, open-label, randomized, parallel-group phase 2 study was conducted in Brazil to further evaluate the safety and efficacy of intravenously administered pabinafusp alfa at 1.0, 2.0, and 4.0 mg/kg/week in MPS-II patients. The safety profiles in the three dosage groups were similar. Neurodevelopmental evaluation suggested positive neurocognitive signals despite a relatively short study period. The 2.0-mg/kg group, which demonstrated marked reductions in substrate concentrations in the CSF, serum, and urine, was considered to provide the best combination regarding safety and efficacy signals.

Mucopolysaccharidosis II (MPS II), a lysosomal storage disease caused by mutations in iduronate-2-sulfatase (IDS), is characterized by a wide variety of somatic and neurologic symptoms. The currently approved intravenous enzyme replacement therapy with recombinant IDS (idursulfase) is ineffective for CNS manifestations due to its inability to cross the blood-brain barrier (BBB). Here, we demonstrate that the clearance of heparan sulfate (HS) deposited in the brain by a BBB-penetrable antibody-enzyme fusion protein prevents neurodegeneration and neurocognitive dysfunctions in MPS II mice. The fusion protein pabinafusp alfa was chronically administered intravenously to MPS II mice. The drug reduced HS and attenuated histopathological changes in the brain, as well as in peripheral tissues. The loss of spatial learning abilities was completely suppressed by pabinafusp alfa, but not by idursulfase, indicating an association between HS deposition in the brain, neurodegeneration, and CNS manifestations in these mice. Furthermore, HS concentrations in the brain and reduction thereof by pabinafusp alpha correlated with those in the cerebrospinal fluid (CSF). Thus, repeated intravenous administration of pabinafusp alfa to MPS II mice decreased HS deposition in the brain, leading to prevention of neurodegeneration and maintenance of neurocognitive function, which may be predicted from HS concentrations in CSF.

Hunter syndrome (mucopolysaccharidosis II [MPS II]), a deficiency of iduronate-2-sulfatase (IDS), causes an accumulation of glycosaminoglycans, giving rise to multiple systemic and CNS symptoms. The currently available therapies, idursulfase and idursulfase beta, are ineffective against the CNS symptoms because they cannot pass the blood-brain barrier (BBB). A novel IDS fused with anti-human transferrin receptor antibody (JR-141) has been shown to penetrate the BBB and ameliorate learning deficits in model mice. This first-in-human study evaluated the pharmacokinetics, safety, and potential efficacy of JR-141 in 14 patients with MPS II. In a dose-escalation study performed in two patients, JR-141 plasma concentrations were dose dependent and peaked at 3 hr after initiation of each infusion, and no or only mild adverse reactions were exhibited. In a subsequent 4-week evaluation at two dose levels, the plasma concentration profiles were similar between the first and final administration, indicating no drug accumulation. Levels of heparan sulfate (HS) and dermatan sulfate (DS) were suppressed in both plasma and urine and HS levels were significantly decreased in cerebrospinal fluid. Two patients experienced some amelioration of neurocognitive and motor symptoms. These results suggest that the drug successfully penetrates the BBB and could have CNS efficacy., Okuyama et al. report on a first-in-human study of iduronate-2-sulfatase fused with anti-human transferrin receptor antibody (JR-141) in 14 patients with mucopolysaccharidosis II to evaluate its pharmacokinetics, safety, and potential efficacy. The results show successful penetration of the compound across the blood-brain barrier, suggestive of its effect for neurodegeneration.

Although congenital scoliosis is defined as a genetic disease characterized by a congenital and abnormal curvature of the spinal vertebrae, our knowledge of the genetic underpinnings of the disease is insufficient. We herein show that the downregulation of the retinol-retinoic acid metabolism pathway is involved in the pathogenesis of congenital scoliosis. By analyzing DNA microarray data, we found that the expression levels of genes associated with the retinol metabolism pathway were decreased in the lumbar spine of Ishibashi rats (IS), a rat model of congenital kyphoscoliosis. The expression of Adh1 and Aldh1a2 (alcohol dehydrogenase), two enzymes that convert retinol to retinoic acid in this pathway, were decreased at both the gene and protein levels. Rarα, a receptor of retinoic acid and bone morphogenetic protein 2, which play a central role in bone formation and are located downstream of this pathway, were also downregulated. Interestingly, the serum retinol levels of IS rats were higher than those of wild-type control rats. These results indicate that the adequate conversion from retinol to retinoic acid is extremely important in the regulation of normal bone formation and it may also be a key factor for understanding the pathogenesis of congenital scoliosis.

Background: Hunter syndrome (mucopolysaccharidosis II, MPS II) is an X-linked recessive lysosomal storage disease in which a deficiency of iduronate-2-sulfatase causes an accumulation of glycosaminoglycans, giving rise to multiple systemic as well as central nervous system (CNS) symptoms. The currently available enzyme replacement therapy with idursulfase is ineffective against the associated CNS symptoms, because the blood-brain barrier (BBB) prevents the enzyme from reaching the brain. A novel iduronate-2-sulfatase fused with anti-human transferrin receptor antibody (JR-141) has been shown to penetrate the BBB and ameliorate learning deficits in model mice. This first-in-human translational study evaluated the pharmacokinetics, safety and efficacy of JR-141 in patients with MPS II. Methods: In this phase 1/2 multicentre, open-label, randomised clinical trial, 15 patients with MPS II who were being treated with intravenous idursulfase were recruited in Japan, and treatment was switched to intravenous administration of JR-141. In Part 1 of the study, which was designed to evaluate the safety of JR-141, the dosage was escalated over a period of 4 weeks (from 0.01 to 2.0 mg/kg/week); in Part 2, a 4-week randomised evaluation of the pharmacokinetics, safety and exploratory efficacy of JR-141 (at 1.0 or 2.0 mg/kg/week) was made. Cerebrospinal fluid (CSF) concentrations of heparan sulfate (HS) and dermatan sulfate (DS) were compared at the start of JR-141 administration and after 3 weeks, and plasma and urine concentrations of HS and DS were compared at the start and after 4 weeks. Findings: Between 10th May, 2017 and 25th October, 2017, we recruited a total of 15 male patients from 9 investigational sites in Japan. Of these, 14 were enrolled after 1 was excluded as unsuitable. Two patients without intellectual disabilities were selected for the dose escalation study (Part 1) and were given 0*01, 0*1, 1.0, and 2*0 mg/kg/week of JR-141 intravenously; plasma concentrations of JR-141 were dose-dependent and peaked at 3 h after initiation of each infusion. One patient developed erythema and pyrexia, which abated quickly, and the other exhibited no adverse reactions. In Part 2, the remaining 12 patients were randomly allocated to JR-141 dosing groups of 1*0 or 2*0 mg/kg/week. The plasma concentration profiles of JR-141 did not differ between the first and final administration, indicating no drug accumulation. Plasma and urinary levels of HS and DS were both suppressed by JR-141, and HS concentrations in the CSF had significantly decreased after the last administration (by an average of 25*1% in patients taking 1*0 mg/kg/week, and 31*5% in those taking 2*0 mg/kg/week). Two patients showed some amelioration of neurocognitive and motor symptoms following the 4-week treatment. No drug-related serious adverse events were observed during the study period. Interpretation: Patients with Hunter syndrome who were treated with JR-141 showed significant reductions in neurotoxin levels in the CSF, suggesting the drug successfully penetrated the BBB. Furthermore, JR-141 was no less effective in treating systemic symptoms than conventional enzyme replacement therapy with idursulfase. If JR-141 reduces glycosaminoglycan accumulation in the CSF, it should also be effective in treating CNS symptoms. This will be investigated in future clinical trials. Clinical Trial Number: NCT03128593 Funding Statement: The trial was funded by JCR Pharmaceuticals. Declaration of Interests: Torayuki Okuyama reports research grants from Sanofi, JCR Pharmaceuticals, GC Pharma, Dainippon Sumitomo Pharma, Actelion, Alexion and Anges, along with honoraria from Sanofi, Dainippon Sumitomo Pharma, Actelion and Anges. Yoshikatsu Eto has conducted consultancy for JCR Pharmaceuticals, and he has been awarded grants and research support from Actelion, BioMarin Pharmaceutical Inc. and Sanofi; he has also received honoraria from Actelion, BioMarin Pharmaceutical Inc., Sanofi, Shire and Dainippon Sumitomo Pharma. Norio Sakai has conducted consultancy for JCR Pharmaceuticals, and he has been awarded grant/research support from Sanofi and Dainippon Sumitomo Pharma, and honoraria from Actelion, BioMarin Pharmaceutical Inc., Sanofi, Shire and Dainippon Sumitomo Pharma. Ethics Approval Statement: The study was conducted in 8 hospitals and specialised MPS centres in Japan, and was in compliance with the Declaration of Helsinki. The protocol and patient informed consent were reviewed and approved by the Institutional Review Board at each participating institution.

We describe the secretory production of a mouse anti-bovine ribonuclease A single-chain variable fragment (scFv) fused with the Fc region of human IgG1 in stably transformed lepidopteran insect cells. Use of the insect-derived Bip and melittin signal peptides resulted in higher yields of the secreted scFv–Fc fusion protein than use of the baculovirus gp64 signal peptide. After cotransfection with an expression vector that contains the Bip signal sequence upstream of the DNA encoding the scFv–Fc and a selection vector that carries a neomycin resistance gene, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were cultured in the presence of G418. Colonies of resistant cells were obtained around 2 weeks after adding G418, and clonal cells were screened by enzyme-linked immunosorbent assay (ELISA) of the culture supernatant. The yield of the scFv–Fc protein secreted from the most productive clone was around 60 mg/l in a shake-flask culture. To improve the productivity, we investigated the effect of medium supplements of sodium butyrate (NaBu), dimethyl sulfoxide (DMSO), and sericin. Supplementing culture medium with sericin increased the scFv–Fc protein yield to 82 mg/l, but productivity was not increased by either NaBu or DMSO. These results indicate that the stably transformed insect cells may allow for the efficient production of scFv–Fc and other Fc fusion proteins.

The effects of cytoplasmic and periplasmic chaperones on the secretory production of an anti-bovine ribonuclease A single-chain variable fragment (scFv) 3A21 in Escherichia coli were investigated. Co-expression of a cytoplasmic chaperone, GroEL/ES, DnaK/DnaJ/GrpE, trigger factor, or SecB with 3A21 scFv affected the proportions of antigen-binding activity in the cytoplasmic soluble fraction, the periplasmic fraction, and the extracellular medium, but there was no significant difference in the total activity compared to the control without chaperone co-expression. On the other hand, co-expression of a periplasmic chaperone, Skp or FkpA, with the exception of DsbC, greatly increased the binding activity in all the soluble fractions. Co-expression of both Skp and FkpA had no synergistic effect. Combinations of cytoplasmic and periplasmic chaperones decreased the productivity. In shake-flask cultures of cells co-expressing Skp or FkpA, considerable amounts of 3A21 scFv were detected in the extracellular medium by enzyme-linked immunosorbent assay (ELISA) and Western blot, and the extracellular production level of 3A21 scFv was calculated to be around 40 mg/l. The binding activity of 3A21 scFv co-expressed with Skp was slightly higher than that with FkpA. These results indicate that the co-expression of periplasmic chaperones Skp and FkpA is extremely useful for the secretory production of scFvs in a culture medium using E. coli, but cytoplasmic chaperones and multiple-chaperone combinations may not be effective.

We describe the soluble production of a mouse anti-bovine ribonuclease A single-chain variable fragment (scFv) fused with the Fc region of human IgG1 in Escherichia coli. Two production systems, secretory production using a pelB signal peptide and cytoplasmic production in a trxB/gor double mutant strain with an oxidizing cytoplasm, were investigated for efficient production of soluble and functional scFv-Fc fusion protein. Antigen-binding activity was observed in both systems but almost all of the scFv-Fc that was expressed formed insoluble aggregates. Hence, the co-expression of molecular chaperones was examined. Co-expression of GroEL/GroES showed a 4.6-fold increase in antigen-binding activity in the cytoplasmic production system but not in the secretory system. By contrast, the other two chaperones, DnaK/DnaJ/GrpE and trigger factor, had no effect in either production system. The protein solubility was also improved markedly by the co-expression of GroEL/GroES and approximately 70% of the 3A21 scFv-Fc protein was soluble. A practical productivity of more than 10 mg/L was achieved with a simple batch shake-flask culture. These results indicate that the E. coli cytoplasmic production system with oxidizing cytoplasm and molecular chaperones might be one of the choices for the soluble production of scFv-Fcs and other Fc fusion proteins.

The Vibrio proteolyticus aminopeptidase is synthesized as a preproprotein and then converted into an active enzyme by cleavage of the N-terminal propeptide. In recombinant Escherichia coli, however, the aminopeptidase is not processed correctly and the less-active form that has the N-terminal propeptide accumulates in the culture medium. Recently, we isolated a novel vibriolysin that was expressed as an active form in E. coli by random mutagenesis; this enzyme shows potential as a candidate enzyme for the processing of aminopeptidase. The E. coli cells were engineered to co-express the novel vibriolysin along with aminopeptidase. Co-expression of vibriolysin resulted in an approximately 13-fold increase in aminopeptidase activity, and a further increase was observed in the form lacking its C-terminal propeptide. The active aminopeptidase was purified from the culture supernatant including the recombinant vibriolysin by heat treatment and ion exchange and hydroxyapatite chromatography with high purity and 35% recovery rate. This purified aminopeptidase effectively converted methionyl-human growth hormone (Met-hGH) to hGH. Thus, this co-expression system provides an efficient method for producing active recombinant V. proteolyticus aminopeptidase.

Vibriolysin, an extracellular protease of Vibrio proteolyticus, is synthesized as a preproenzyme. The N-terminal propeptide functions as an intramolecular chaperone and an inhibitor of the mature enzyme. Extracellular production of recombinant vibriolysin has been achieved in Bacillus subtilis, but not in Escherichia coli, which is widely used as a host for the production of recombinant proteins. Vibriolysin is expressed as an inactive form in E. coli possibly due to the inhibitory effect of the N-terminal propeptide. In this study, we isolated the novel vibriolysin engineered by in vivo random mutagenesis, which is expressed as active mature vibriolysin in E. coli. The Western blot analysis showed that the N-terminal propeptide of the engineered enzyme was processed and degraded, confirming that the propeptide inhibits the mature enzyme. Two mutations located within the engineered vibriolysin resulted in the substitution of stop codon for Trp at position 11 in the signal peptide and of Val for Ala at position 183 in the N-terminal propeptide (where position 1 is defined as the first methionine). It was found that the individual mutations are related to the enzyme activity. The novel vibriolysin was extracellularly overproduced in BL21(DE3) and purified from the culture supernatant by ion-exchange chromatography followed by hydrophobic-interaction chromatography, resulting in an overall yield of 2.2 mg/L of purified protein. This suggests that the novel engineered vibriolysin is useful for overproduction in an E. coli expression system.

Recombinant human growth hormone (r-hGH) overexpressed in Escherichia coli forms inactive and insoluble aggregates as inclusion bodies in the cytoplasm. The efficient solubilization of inclusion bodies is critical for cost-effective production. Contrary to a previous report, in our production system, the solubilization method by alkaline treatment including 2 M urea was ineffective. Hence various buffers containing different concentrations of urea or guanidine hydrochloride (GnHCl) at neutral and alkaline pH were attempted. Efficient solubilization (about 90%) was observed in 100 mM Tris buffer, pH 8.0, with more than 4 M GnHCl, and at pH 12.5 with more than 2 M GnHCl, but not with about 8 M of urea. The r-hGH solubilized at pH 12.5 containing 2 M GnHCl was refolded by simple dilution and purified by DEAE Sepharose anion-exchange chromatography. The biological activity of the resulting r-hGH was comparable with commercially available r-hGH in in vitro cell proliferation assay using the hGH-dependent cell line.

Agrobacterium tumefaciens harboring a Ti plasmid causes crown gall disease in dicot plants by transferring its T-DNA into plant chromosomes. Iron acquisition plays an important role for pathogenicity in animal pathogens and several phytopathogens and for growth in the rhizosphere and on plant surfaces. Under iron-limiting condition, bacteria produce various iron-chelating agents called siderophores. Agrobacterium strains have the diversity in producing siderophores and a certain strain produces a typical catechol-type siderophore, called agrobactin, although its biosynthesis genes have not been analyzed yet. Here we describe the cloning and characterization of a functional gene cluster involved in ferric iron uptake in A. tumefaciens strain MAFF301001. Four complete open reading frames (ORFs) were found in 5-kb region of a genomic library clone 1A3. We named these genes agb, after agrobactin. agbC, agbE, agbB and agbA genes were identified in this order, and narrow intergenic spaces suggested that these genes constitute an operon. Predicted agb gene products and their phylogenetic analysis showed sequence similarity with enzymes which are involved in ferric iron uptake in other bacteria. Southern hybridization analysis clearly indicated the location of agb genes on the linear chromosome in strain MAFF301001 but the complete lack in another A. tumefaciens strain C58. Mutation analysis of agbB revealed that it is essential for growth and production of catechol compounds in iron-limiting medium.