Keisuke Kitakaze

Therapeutic Potential of Intracerebroventricular Replacement of Modified Human β-Hexosaminidase B for GM2 Gangliosidosis

To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of β-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human β-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB.

Chemical Synthesis of Biologically Active Monoglycosylated GM2‐Activator Protein Analogue Using N‐Sulfanylethylanilide Peptide

Going to SEA(lide): Total chemical synthesis of a 162-residue glycoprotein analogue of the monoglycosylated human GM2-activator protein (GM2AP) was achieved. Key steps were the use of N-sulfanylethylanilide (SEAlide) peptides in the kinetic chemical ligation synthesis of a large peptide fragment, and a convergent native chemical ligation for final fragment assembly.

Lyso-GM2 Ganglioside: A Possible Biomarker of Tay-Sachs Disease and Sandhoff Disease

To find a new biomarker of Tay-Sachs disease and Sandhoff disease. The lyso-GM2 ganglioside (lyso-GM2) levels in the brain and plasma in Sandhoff mice were measured by means of high performance liquid chromatography and the effect of a modified hexosaminidase (Hex) B exhibiting Hex A-like activity was examined. Then, the lyso-GM2 concentrations in human plasma samples were determined. The lyso-GM2 levels in the brain and plasma in Sandhoff mice were apparently increased compared with those in wild-type mice, and they decreased on intracerebroventricular administration of the modified Hex B. The lyso-GM2 levels in plasma of patients with Tay-Sachs disease and Sandhoff disease were increased, and the increase in lyso-GM2 was associated with a decrease in Hex A activity. Lyso-GM2 is expected to be a potential biomarker of Tay-Sachs disease and Sandhoff disease.

Protease-resistant modified human β-hexosaminidase B ameliorates symptoms in GM2 gangliosidosis model

GM2 gangliosidoses, including Tay-Sachs and Sandhoff diseases, are neurodegenerative lysosomal storage diseases that are caused by deficiency of β-hexosaminidase A, which comprises an αβ heterodimer. There are no effective treatments for these diseases; however, various strategies aimed at restoring β-hexosaminidase A have been explored. Here, we produced a modified human hexosaminidase subunit β (HexB), which we have termed mod2B, composed of homodimeric β subunits that contain amino acid sequences from the α subunit that confer GM2 ganglioside-degrading activity and protease resistance. We also developed fluorescent probes that allow visualization of endocytosis of mod2B via mannose 6-phosphate receptors and delivery of mod2B to lysosomes in GM2 gangliosidosis models. In addition, we applied imaging mass spectrometry to monitor efficacy of this approach in Sandhoff disease model mice. Following i.c.v. administration, mod2B was widely distributed and reduced accumulation of GM2, asialo-GM2, and bis(monoacylglycero)phosphate in brain regions including the hypothalamus, hippocampus, and cerebellum. Moreover, mod2B administration markedly improved motor dysfunction and a prolonged lifespan in Sandhoff disease mice. Together, the results of our study indicate that mod2B has potential for intracerebrospinal fluid enzyme replacement therapy and should be further explored as a gene therapy for GM2 gangliosidoses.

Development of a Chemical Methodology for the Preparation of Peptide Thioesters Applicable to Naturally Occurring Peptides Using a Sequential Quadruple Acyl Transfer System

Peptide thioesters are very useful in protein chemistry, and chemistry- and biochemistry-based protocols are used for the preparation of thioesters. Among such protocols, only a few biochemistry-based approaches have been use for naturally occurring peptide sequences. The development of chemistry-based protocols applicable to natural sequences remains a challenge, and the development of such methods would be a major contribution to protein science. Here, we describe the preparation of peptide thioesters using innovative methodology that features nickel(II)-mediated alcoholysis of a naturally occurring peptide sequence, followed by O−N and N−S acyl transfers. This protocol involves sequential quadruple acyl transfer, termed SQAT. Notably, the SQAT system consists of sequential chemical reactions that allow naturally occurring peptide sequences to be converted to thioesters without requiring an artificial chemical unit.

Tailored Synthesis of 162-Residue S-Monoglycosylated GM2-Activator Protein (GM2AP) Analogues that Allows Facile Access to a Protein Library.

A synthetic protocol for the preparation of 162‐residue S‐monoglycosylated GM2‐activator protein (GM2AP) analogues bearing various amino acid substitutions for Thr69 has been developed. The facile incorporation of the replacements into the protein was achieved by means of a one‐pot/N‐to‐C‐directed sequential ligation strategy using readily accessible middle N‐sulfanylethylanilide (SEAlide) peptides each consisting of seven amino acid residues. A kinetically controlled ligation protocol was successfully applied to the assembly of three peptide segments covering the GM2AP. The native chemical ligation (NCL) reactivities of the SEAlide peptides can be tuned by the presence or absence of phosphate salts. Furthermore, NCL of the alkyl thioester fragment [GM2AP (1–31)] with the N‐terminal cysteinyl prolyl thioester [GM2AP (32–67)] proceeded smoothly to yield the 67‐residue prolyl thioester, with the prolyl thioester moiety remaining intact. This newly developed strategy enabled the facile synthesis of GM2AP analogues. Thus, we refer to this synthetic protocol as “tailored synthesis” for the construction of a GM2AP library.

Combined replacement effects of human modified β-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts

GM2 gangliosidoses are autosomal recessive lysosomal storage diseases (LSDs) caused by mutations in the HEXA, HEXB and GM2A genes, which encode the human lysosomal β-hexosaminidase (Hex) α- and β-subunits, and GM2 activator protein (GM2A), respectively. These diseases are associated with excessive accumulation of GM2 ganglioside (GM2) in the brains of patients with neurological symptoms. Here we established a CHO cell line overexpressing human GM2A, and purified GM2A from the conditioned medium, which was taken up by fibroblasts derived from a patient with GM2A deficiency, and had the therapeutic effects of reducing the GM2 accumulated in fibroblasts when added to the culture medium. We also demonstrated for the first time that recombinant GM2A could enhance the replacement effect of human modified HexB (modB) with GM2-degrading activity, which is composed of homodimeric altered β-subunits containing a partial amino acid sequence of the α-subunit, including the GSEP loop necessary for binding to GM2A, on reduction of the GM2 accumulated in fibroblasts derived from a patient with Tay-Sachs disease, a HexA (αβ heterodimer) deficiency, caused by HEXA mutations. We predicted the same manner of binding of GM2A to the GSEP loop located in the modified HexB β-subunit to that in the native HexA α-subunit on the basis of the x-ray crystal structures. These findings suggest the effectiveness of combinational replacement therapy involving the human modified HexB and GM2A for GM2 gangliosidoses.

Development of Chemistry-Based Protocol for Sequence-Dependent Thioesterification

One of the useful methods for exploration of the function of proteins is utilization of the synthesized proteins which include artificial units such as fluorescence dye. For the synthesis of such proteins, a peptide thioester and an N-terminal cysteinyl peptide prepared by Solid Phase Peptide Synthesis (SPPS) are coupled to each other using Native Chemical Ligation (NCL) [1]. However, due to the limitation of the number of amino acid residues applicable to SPPS, multistep NCLs are required for chemical synthesis of large proteins. On the other hand, in semi-synthesis of proteins, only single NCL of protein thioester prepared from expressed protein with chemically synthesized N-terminal cysteinyl peptide enables to incorporate functional moieties to large proteins. However, there are only a few methods for preparation of protein thioesters applicable to naturally occurring sequences [2]. Therefore, new protocols for preparation of protein thioesters has been required.