16 research outputs found
spERt Technology: A novel strategy to improve productivity through enhanced polyribosome assembly on the endoplasmic reticulum in CHO cells
In cell line development process, it is frequently observed that increased mRNA levels do not always correlate with protein expression levels in CHO cells. In line with this gap, the endoplasmic reticulum (ER) in CHO cells is much less proliferated as compared with that in terminally differentiated (i.e., professional) secretory cells, suggesting that there is still room to improve their specific productivity if translational efficiency on the ER can be up-regulated. Here we present a novel engineering approach (spERt Technology) to improve specific production rates by mimicking the ER translational apparatus of professional secretory cells. In spERt Technology, we exploit the unique factors that are required for translationally active polyribosome formation on the ER to directly enhance the translational efficiency (1, 2). A high antibody (Ab) producing clone generated by a novel screen using flow cytometry (3) was used as a model cell line. The factors were introduced into the high producer and a series of the spERt Technology - introduced cell lines were generated Among these cell lines, we selected one of the best clones (spERt-f9) having stable and high productivity. Polyribosome analysis of these cell lines revealed that enhanced assembly of the ER polyribosomes as expected (1). Consistent with the highly developed polyribosomes, the spERt-introduced cell lines produced higher levels of Ab than that of parental cells, and showed prominent increase of specific production rates. Further optimization of feeding process resulted in remarkable increase of productivity in spERt-f9 cells: Ab titers of 7.6 g/L and 9.5 g/L on day 14 and 17, respectively, were achieved in shake flask fed-batch cultures by using chemically defined media. Importantly, high cell viabilities were maintained in spERt-f9 cells throughout the culture periods. In addition, lower glucose consumption and reduced accumulation of ammonia were observed. Product quality in these cells were analyzed and compared with that in the parental cells. In conclusion, spERt Technology enables to improve productivity of high Ab producers, associated with reduced accumulation of waste metabolites and high cell viabilities
Stable Isotope-Labeled Collagen: A Novel and Versatile Tool for Quantitative Collagen Analyses Using Mass Spectrometry
Collagens
are the most abundant proteins in animals and are involved
in many physiological/pathological events. Although various methods
have been used to quantify collagen and its post-translational modifications
(PTMs) over the years, it is still difficult to accurately quantify
type-specific collagen and minor collagen PTMs. We report a novel
quantitative method targeting collagen using stable isotope-labeled
collagen named “SI-collagen”, which was labeled with
isotopically heavy lysine, arginine, and proline in fibroblasts culture.
We prepared highly labeled and purified SI-collagen for use as an
internal standard in mass spectrometric analysis, particularly for
a new approach using amino acid hydrolysis. Our method enabled accurate
collagen analyses, including quantification of (1) type-specific collagen
(types I and III in this paper), (2) total collagen, and (3) collagen
PTMs by LC–MS with high sensitivity. SI-collagen is also applicable
to other diverse analyses of collagen and can be a powerful tool for
various studies, such as detailed investigation of collagen-related
disorders
Highly Accurate Quantification of Hydroxyproline-Containing Peptides in Blood Using a Protease Digest of Stable Isotope-Labeled Collagen
Collagen-derived
hydroxyproline (Hyp)-containing dipeptides and
tripeptides, which are known to possess physiological functions, appear
in blood at high concentrations after oral ingestion of gelatin hydrolysate.
However, highly accurate and sensitive quantification of the Hyp-containing
peptides in blood has been challenging because of the analytical interference
from numerous other blood components. We recently developed a stable
isotope-labeled collagen named “SI-collagen” that can
be used as an internal standard in various types of collagen analyses
employing liquid chromatography–mass spectrometry (LC-MS).
Here we prepared stable isotope-labeled Hyp-containing peptides from
SI-collagen using trypsin/chymotrypsin and plasma proteases by mimicking
the protein degradation pathways in the body. With the protease digest
of SI-collagen used as an internal standard mixture, we achieved highly
accurate simultaneous quantification of Hyp and 13 Hyp-containing
peptides in human blood by LC-MS. The area under the plasma concentration–time
curve of Hyp-containing peptides ranged from 0.663 ± 0.022 nmol/mL·h
for Pro-Hyp-Gly to 163 ± 1 nmol/mL·h for Pro-Hyp after oral
ingestion of 25 g of fish gelatin hydrolysate, and the coefficient
of variation of three separate measurements was <7% for each peptide
except for Glu-Hyp-Gly, which was near the detection limit. Our method
is useful for absorption/metabolism studies of the Hyp-containing
peptides and development of functionally characterized gelatin hydrolysate
The Transcription Factor HAND1 Is Involved in Cortical Bone Mass through the Regulation of Collagen Expression
Temporal and/or spatial alteration of collagen family gene expression results in bone defects. However, how collagen expression controls bone size remains largely unknown. The basic helix-loop-helix transcription factor HAND1 is expressed in developing long bones and is involved in their morphogenesis. To understand the functional role of HAND1 and collagen in the postnatal development of long bones, we overexpressed Hand1 in the osteochondroprogenitors of model mice and found that the bone volumes of cortical bones decreased in Hand1Tg/+;Twist2-Cre mice. Continuous Hand1 expression downregulated the gene expression of type I, V, and XI collagen in the diaphyses of long bones and was associated with decreased expression of Runx2 and Sp7/Osterix, encoding transcription factors involved in the transactivation of fibril-forming collagen genes. Members of the microRNA-196 family, which target the 3′ untranslated regions of COL1A1 and COL1A2, were significantly upregulated in Hand1Tg/+;Twist2-Cre mice. Mass spectrometry revealed that the expression ratios of alpha 1(XI), alpha 2(XI), and alpha 2(V) in the diaphysis increased during postnatal development in wild-type mice, which was delayed in Hand1Tg/+;Twist2-Cre mice. Our results demonstrate that HAND1 regulates bone size and morphology through osteochondroprogenitors, at least partially by suppressing postnatal expression of collagen fibrils in the cortical bones
Efficient Absorption of X‑Hydroxyproline (Hyp)-Gly after Oral Administration of a Novel Gelatin Hydrolysate Prepared Using Ginger Protease
Recent studies have reported that
oral intake of gelatin hydrolysate
has various beneficial effects, such as reduction of joint pain and
lowering of blood sugar levels. In this study, we produced a novel
gelatin hydrolysate using a cysteine-type ginger protease having unique
substrate specificity with preferential peptide cleavage with Pro
at the P<sub>2</sub> position. Substantial amounts of X-hydroxyproline
(Hyp)-Gly-type tripeptides were generated up to 2.5% (w/w) concomitantly
with Gly-Pro-Y-type tripeptides (5%; w/w) using ginger powder. The
in vivo absorption of the ginger-degraded gelatin hydrolysate was
estimated using mice. The plasma levels of collagen-derived oligopeptides,
especially X-Hyp-Gly, were significantly high (e.g., 2.3-fold for
Glu-Hyp-Gly, <i>p</i> < 0.05) compared with those of
the control gelatin hydrolysate, which was prepared using gastrointestinal
proteases and did not contain detectable X-Hyp-Gly. This study demonstrated
that orally administered X-Hyp-Gly was effectively absorbed into the
blood, probably due to the high protease resistance of this type of
tripeptide
Identification of Collagen-Derived Hydroxyproline (Hyp)-Containing Cyclic Dipeptides with High Oral Bioavailability: Efficient Formation of Cyclo(X-Hyp) from X‑Hyp-Gly-Type Tripeptides by Heating
Cyclic
dipeptides (2,5-diketopiperazines) are present in a variety
of foods and are reported to demonstrate antioxidant, antidepressant,
and other beneficial effects. We recently developed a novel collagen
hydrolysate characterized by a high content of X-hydroxyproline (Hyp)-Gly-type
tripeptides using ginger protease. In the present study, we found
that, through heating, X-Hyp-Gly can be easily converted into Hyp-containing
cyclic dipeptides. After heating for 3 h at 85 °C and pH 4.8,
Ala-Hyp-Gly was almost completely cyclized to cycloÂ(Ala-Hyp), in contrast
to a slight cyclization of Ala-Hyp. The contents of cycloÂ(Ala-Hyp)
and cycloÂ(Leu-Hyp) reached 0.5–1% (w/w) each in the ginger-degraded
collagen hydrolysate under the heating conditions. Oral administration
experiments using mice revealed that cycloÂ(Ala-Hyp) and cycloÂ(Leu-Hyp)
were absorbed into the blood at markedly higher efficiencies compared
to collagenous oligopeptides, including Pro-Hyp. The high productivity
and oral bioavailability of the collagen-specific cyclic dipeptides
suggest significant health benefits of the heat-treated ginger-degraded
collagen hydrolysate
Production of a novel wheat gluten hydrolysate containing dipeptidyl peptidase-IV inhibitory tripeptides using ginger protease
<p>Wheat gluten is a Pro-rich protein complex comprising glutenins and gliadins. Previous studies have reported that oral intake of enzymatic hydrolysates of gluten has beneficial effects, such as suppression of muscle injury and improvement of hepatitis. Here, we utilized ginger protease that preferentially cleaves peptide bonds with Pro at the P<sub>2</sub> position to produce a novel type of wheat gluten hydrolysate. Ginger protease efficiently hydrolyzed gluten, particularly under weak acidic conditions, to peptides with an average molecular weight of <600 Da. In addition, the gluten hydrolysate contained substantial amounts of tripeptides, including Gln-Pro-Gln, Gln-Pro-Gly, Gln-Pro-Phe, Leu-Pro-Gln, and Ser-Pro-Gln (e.g. 40.7 mg/g at pH 5.2). These gluten-derived tripeptides showed high inhibitory activity on dipeptidyl peptidase-IV with IC<sub>50</sub> values of 79.8, 70.9, 71.7, 56.7, and 78.9 μM, respectively, suggesting that the novel gluten hydrolysate prepared using ginger protease can be used as a functional food for patients with type 2 diabetes.</p> <p>Production of DPP-IV inhibitory tripeptides from gluten using ginger protease.</p
IgA polymerization contributes to efficient virus neutralization on human upper respiratory mucosa after intranasal inactivated influenza vaccine administration
Unlike the current injectable influenza vaccines, intranasally administered influenza vaccines induce influenza virus-specific IgA antibodies in the local respiratory mucosa as well as IgG antibodies in the systemic circulation. Our previous study showed that after five volunteers underwent intranasal administration with inactivated H3N2 or H5N1 vaccines, their IgA antibodies on the upper respiratory tract were present as monomers, dimers, and multimers (trimers and tetramers). Moreover, the multimers associated with the highest virus neutralizing activity. However, it has remained elusive whether a more practical intranasal vaccination strategy could induce the high-performance IgA multimers in the nasal mucosa. In the present study, volunteers were administered with two doses of the intranasal trivalent whole-virus inactivated influenza vaccine and showed that in nasal wash samples the amount of multimeric IgA correlated positively with virus neutralizing titers, indicating that the multimeric IgA antibodies play an important role in the antiviral activity at the nasal mucosa. Surface plasmon resonance analysis of the binding dynamics of nasal wash derived IgA monomers, dimers, and multimers against recombinant trimeric influenza virus HA showed that sample fractions containing IgA multimers dissociated from HA less well than sample fractions without IgA multimers. Thus, IgA multimers may “stick” to the antigen more tightly than the other structures. In summary, intranasal administration of two doses of multivalent inactivated influenza vaccines induced multimeric IgA. Multimerization of mucosal IgA antibodies conferred higher neutralizing activity against viruses in the nasal mucosa, possibly by increasing their cohesion to virus antigens