Alteogen (South Korea)

PH-20 is a specific type of hyaluronidase that plays a critical role in the fertilization process by facilitating the initial binding of sperm to the glycoprotein layer surrounding the oocyte and subsequently breaking down hyaluronic acid polymers in the cumulus cell layer. PH-20 contains an epidermal growth factor (EGF)-like domain, which may be involved in the recognition of the glycoprotein layer in addition to the catalytic domain. Herein, we report the structure of human PH-20 determined by cryogenic electron microscopy. Comparative analyses of the PH-20 structure with two other available hyaluronidase structures reveal a general similarity in the central catalytic domains, including the conservation of catalytically essential residues at the equivalent spatial positions. However, unique difference is found in the EGF-like domain, characterized by a longer sequence that is likely to form a flexibly anchored β-hairpin containing a disulfide bond.

Here, we demonstrate that a metal ion binding motif could serve as an efficient and robust tool for site-specific conjugation strategy. Cysteine-containing metal binding motifs were constructed as single repeat or tandem repeat peptides and their metal binding characteristics were investigated. The tandem repeats of the Cysteine-Glycine-Histidine (CGH) metal ion binding motif exhibited concerted binding to Co(II) ions, suggesting that conformational transition of peptide was triggered by the sequential metal ion binding. Evaluation of the free thiol content after reduction by reducing reagent showed that metal-ion binding elicited strong retardation of cysteine oxidation in the order of Zn(II)>Ni(II)>Co(II). The CGH metal ion binding motif was then introduced to the C-terminus of antibody heavy chain and the metal ion-dependent characteristics of oxidation kinetics were investigated. As in the case of peptides, CGH-motif-introduced antibody exhibited strong dependence on metal ion binding to protect against oxidation. Zn(II)-saturated antibody with tandem repeat of CGH motif retains the cysteine reactivity as long as 22 hour even with saturating O2 condition. Metal-ion dependent fluorophore labeling clearly indicated that metal binding motifs could be employed as an efficient tool for site-specific conjugation. Whereas Trastuzumab without a metal ion binding site exhibited site-nonspecific dye conjugation, Zn(II) ion binding to antibody with a tandem repeat of CGH motif showed that fluorophores were site-specifically conjugated to the heavy chain of antibody. We believe that this strong metal ion dependence on oxidation protection and the resulting site-selective conjugation could be exploited further to develop a highly site-specific conjugation strategy for proteins that contain multiple intrinsic cysteine residues, including monoclonal antibodies.

Human alpha-1-antitrypsin (α1AT) is a glycoprotein with protease inhibitor activity protecting tissues from degradation. Patients with inherited α1AT deficiency are treated with native α1AT (nAT) purified from human plasma. In the present study, recombinant α1AT (rAT) was produced in Chinese hamster ovary (CHO) cells and their glycosylation patterns, inhibitory activity and in vivo half-life were compared with those of nAT. A peptide mapping analysis employing a deglycosylation reaction confirmed full occupancy of all three glycosylation sites and the equivalency of rAT and nAT in terms of the protein level. N-glycan profiles revealed that rAT contained 10 glycan structures ranging from bi-antennary to tetra-antennary complex-type glycans while nAT displayed six peaks comprising majorly bi-antennary glycans and a small portion of tri-antennary glycans. In addition, most of the rAT glycans were shown to have only core α(1 - 6)-fucose without terminal fucosylation, whereas only minor portions of the nAT glycans contained core or Lewis X-type fucose. As expected, all sialylated glycans of rAT were found to have α(2 - 3)-linked sialic acids, which was in sharp contrast to those of nAT, which had mostly α(2 - 6)-linked sialic acids. However, the degree of sialylation of rAT was comparable to that of nAT, which was also supported by an isoelectric focusing gel analysis. Despite the differences in the glycosylation patterns, both α1ATs showed nearly equivalent inhibitory activity in enzyme assays and serum half-lives in a pharmacokinetic experiment. These results suggest that rAT produced in CHO cells would be a good alternative to nAT derived from human plasma.

Recently, a novel risk gene protein expressed in elderly patients with the diagnosis of Alzheimer disease (AD) was discovered on chromosome 21 within the APP (amyloid precursor protein) region. This 79 amino acid protein, ALZAS (Alzheimer Associated Protein) contains the beta-amyloid peptide 1-42 fragment, the APP transmembrane signal, and a unique 12 amino acid c-terminal which is not present in any known allele of the APP gene. Reverse transcription-PCR revealed that the transcript of ALZAS was expressed in cortical and hippocampal regions of human Alzheimer disease brain as well as in leukocytes derived from AD patients. Most specifically, an endogenous antibody was found in patients with confirmed AD, in patients with depression, and in subjects suggested to have presymptomatic AD, where it was directed against epitopes within the intron encoded amino acid c-terminal sequence.