Epitope-Based Chicken-Derived Novel Anti-PAD2 Monoclonal Antibodies Inhibit Citrullination.

The aberrant upregulation of protein arginine deiminase 2- (PAD2-) catalyzed citrullination is reported in various autoimmune diseases (rheumatoid arthritis and multiple sclerosis) and several cancers. Currently, there are no anti-PAD2 monoclonal antibodies (mAbs) that can inhibit the citrullination reaction. Here, an epitope 341YLNRGDRWIQDEIEFGY357 was examined as an antigenic site of PAD2. Chickens were immunized with this epitope, and the generated mAbs were screened for its reactivity against the full-length PAD2. Enzyme-linked immunosorbent assay revealed that six mAbs, which were screened from the phage display library, crossreacted with mouse PAD2. Kinetic analysis revealed that mAbs are bound to PAD2 in the nanomolar range, which indicated a strong binding. Results of the in vitro citrullination inhibition assay revealed that the half-maximal effective concentration values of mAbs for the inhibition of histone or benzoyl-L-arginine ethyl ester citrullination were in the range of 6-75 nM which supports strong inhibition capabilities. Alanine scanning of epitope revealed that the peptide fragment 344RGDRWIQDEIEF355 was responsible for generating strong antibody responses that inhibit the PAD2-catalyzed citrullination reaction. These antibodies can aid in understanding the extracellular PAD2 function and treating diseases associated with aberrant citrullination.

Accumulation of foreign polypeptides to rice seed protein body type I using prolamin portion sequences.

KEY MESSAGE: Rice prolamins are accumulated in endoplasmic reticulum (ER)-derived proteins bodies, although conserved sequences retained in ER are not confirmed. We investigated portion sequences of prolamins that must accumulate in PB-Is. Rice seed prolamins are accumulated in endoplasmic reticulum (ER)-derived protein body type I (PB-I), but ER retention sequences in rice prolamin polypeptides have not been confirmed. Here we investigated the lengths of the prolamin portion sequences required for accumulation in PB-Is. Of the rice prolamins, we compared 13a and 13b prolamins because the amino acid sequences of these prolamins are quite similar except for the presence or absence of Cys-residues. We also generated and analyzed transgenic rice expressing several prolamin portion sequence-GFP fusion proteins. We observed that in 13a prolamin, when the portion sequences were extended more than the 68th amino acid residue from the initiating methionine, the prolamin portion sequence-GFP fusion proteins were accumulated in PB-Is. In 13b prolamin, when the portion sequences were extended by more than the 82nd amino acid residue from the initiating methionine, the prolamin portion sequence-GFP fusion proteins were accumulated in PB-Is. When those fusion proteins were extracted under non-reduced or reduced conditions, the 13a prolamin portion sequence-GFP fusion proteins in PB-Is were soluble under only the reduced condition. In contrast, 13b prolamin portion sequence-GFP fusion proteins were soluble under both non-reduced and reduced conditions. These results suggest that the accumulation of 13a prolamin in PB-Is is associated with the formation of disulfide bonds and/or hydrophobicity in 13a prolamin polypeptide, whereas the accumulation of 13b prolamin in PB-Is was less involved in the formation of disulfide bonds.

Control of foreign polypeptide localization in specific layers of protein body type I in rice seed.

KEY MESSAGE: Prolamin-GFP fusion proteins, expressed under the control of native prolamin promoters, were localized in specific layers of PB-Is. Prolamin-GFP fusion proteins were gradually digested from outside by pepsin digestion. In rice seed endosperm, protein body type I (PB-I) has a layered structure consisting of prolamin species and is the resistant to digestive juices in the intestinal tract. We propose the utilization of PB-Is as an oral vaccine carrier to induce mucosal immune response effectively. If vaccine antigens are localized in a specific layer within PB-Is, they could be protected from gastric juice and be delivered intact to the small intestine. We observed the localization of GFP fluorescence in transgenic rice endosperm expressing prolamin-GFP fusion proteins with native prolamin promoters, and we confirmed that the foreign proteins were located in specific layers of PB-Is artificially. Each prolamin-GFP fusion protein was localized in specific layers of PB-Is, such as the outer-most layer, middle layer, and core region. Furthermore, to investigate the resistance of prolamin-GFP fusion proteins against pepsin digestion, we performed in vitro pepsin treatment. Prolamin-GFP fusion proteins were gradually digested from the peripheral region and the contours of PB-Is were made rough by in vitro pepsin treatment. These findings suggested that prolamin-GFP fusion proteins accumulating specific layers of PB-Is were gradually digested and exposed from the outside by pepsin digestion.

Identification of the region of rice 13 kDa prolamin essential for the formation of ER-derived protein bodies using a heterologous expression system.

Cereal prolamins, which are alcohol-soluble seed storage proteins, can induce ER-derived protein bodies (PBs) in heterologous tissue. Like maize and wheat prolamins, rice prolamins can form ER-derived PBs, but the region of mature polypeptides that is essential for PB formation has not been identified. In this study, we examined the formation mechanisms of ER-derived PB-like structures by expressing rice 13 kDa prolamin-deletion mutants fused to green fluorescent protein (GFP) in heterologous tissues such as yeast. The 13 kDa prolamin-GFP fusion protein was stably accumulated in transgenic yeast and formed an ER-derived PB-like structure. In contrast, rice α-globulin-GFP fusion protein was transported to vacuoles. In addition, the middle and COOH-terminal regions of 13 kDa prolamin formed ER-derived PB-like structures, whereas the NH2-terminal region of 13 kDa prolamin did not form such structures. These results suggest that the middle and COOH-terminal regions of 13 kDa prolamin can be retained and thus can induce ER-derived PB in yeast.

Accumulation of rice prolamin-GFP fusion proteins induces ER-derived protein bodies in transgenic rice calli.

KEY MESSAGE : We showed that rice prolamin polypeptides formed ER-derived PBs in transgenic rice calli, and that this heterologous transgene expression system is suitable for studying the mechanism of rice PB-I formation. Rice prolamins, alcohol-soluble seed storage proteins, accumulate directly within the rough endoplasmic reticulum (ER) lumen, leading to the formation of ER-derived type I protein bodies (PB-Is) in rice seed. Because rice prolamins do not possess a well-known ER retention signal such as K(H)DEL, or a unique sequence for retention in the ER such as a tandem repeat domain of maize and wheat prolamins, the mechanisms of prolamin accumulation in the ER and PB-I formation are poorly understood. In this study, we examined the formation mechanisms of PBs by expressing four types of rice prolamin species fused to green fluorescent protein (GFP) in transgenic rice calli. Each prolamin-GFP fusion protein was stably accumulated in rice calli and formed ER-derived PBs. In contrast, GFP fused with the signal peptide of prolamin was secreted into the intercellular space in rice calli. In addition, each of the four types of prolamin-GFP fusion proteins was co-localized with the ER chaperone binding protein. These results suggest that the mature polypeptide of prolamin is capable of being retained in the ER and induce the formation of PBs in non-seed tissue, and that the rice callus heterologous transgene expression system is useful for studying the mechanisms of rice PB-I formation.

Separation and identification of rice prolamins by two-dimensional gel electrophoresis and amino acid sequencing.

There are difficulties in detecting and separating rice prolamin polypeptides by 2D-PAGE analysis because prolamin polypeptides are insoluble, and the amino acid sequences show high homology among them. In this study, we improved the prolamin extraction method and the 2D-PAGE procedure, and succeeded in separating prolamin polypeptide species by 2D-PAGE and in identifying major prolamin polypeptide sequences.

Formation mechanism of the internal structure of type I protein bodies in rice endosperm: relationship between the localization of prolamin species and the expression of individual genes.

Rice prolamins, a group of seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and form type I protein bodies (PB-Is) in endosperm cells. Rice prolamins are encoded by a multigene family. In this study, the spatial accumulation patterns of various prolamin species in rice endosperm cells were investigated to determine the mechanism of formation of the internal structure of PB-Is. Immunofluorescence microscopic analysis of mature endosperm cells showed that the 10 kDa prolamin is mainly localized in the core of the PB-Is, the 13b prolamin is localized in the inner layer surrounding the core and the outermost layer, and the 13a and 16 kDa prolamins are localized in the middle layer. Real-time RT-PCR analysis showed that expression of the mRNA for 10 kDa prolamin precedes expression of 13a, 13b-1 and 16 kDa prolamin in the developing stages. mRNA expression for 13b-2 prolamin occurred after that of the other prolamin species. Immunoelectron microscopy of developing seeds showed that the 10 kDa prolamin polypeptide initially accumulates in the ER, and then 13b, 13a, 16 kDa and 13b prolamins are stacked in layers within the ER. Studies with transgenic rice seeds expressing prolamin-GFP fusion proteins under the control of native and constitutive promoters indicated that the temporal expression pattern of prolamin genes influenced the localization of prolamin proteins within the PB-Is. These findings indicate that the control of gene expression of prolamin species contributes to the internal structure of PB-Is.

Production of human growth hormone in transgenic rice seeds: co-introduction of RNA interference cassette for suppressing the gene expression of endogenous storage proteins.

Rice seeds are potentially useful hosts for the production of pharmaceutical proteins. However, low yields of recombinant proteins have been observed in many cases because recombinant proteins compete with endogenous storage proteins. Therefore, we attempt to suppress endogenous seed storage proteins by RNA interference (RNAi) to develop rice seeds as a more efficient protein expression system. In this study, human growth hormone (hGH) was expressed in transgenic rice seeds using an endosperm-specific promoter from a 10 kDa rice prolamin gene. In addition, an RNAi cassette for reduction of endogenous storage protein expressions was inserted into the hGH expression construct. Using this system, the expression levels of 13 kDa prolamin and glutelin were effectively suppressed and hGH polypeptides accumulated to 470 µg/g dry weight at the maximum level in transgenic rice seeds. These results suggest that the suppression of endogenous protein gene expression by RNAi could be of great utility for increasing transgene products.