Biomedical subjects

Akiko Hasegawa

Publications and source records attributed to Akiko Hasegawa.

At least 19 recordsLinked to original sources

PubMed2006

Regulation of glial development by cystatin C.

Cystatin C (CysC) is an endogenous cysteine proteases inhibitor produced by mature astrocytes in the adult brain. Previously we isolated CysC as a factor activating the glial fibrillary acidic protein (GFAP) promoter, and showed that CysC is expressed in astrocyte progenitors during development. Here we show that protease inhibitor activity increased daily in conditioned medium, and that this activity was mainly a result of CysC released from primary cultured cells. Human CysC added to the culture medium of primary brain cells increased the number of GFAP-positive and nestin-positive cells. Human CysC also increased the number of neurospheres formed from embryonic brain, and thus it increases the number of neural stem/precursor cells in a manner similar to glycosylated rat CysC. The addition of a neutralizing antibody, on the other hand, greatly decreased the number of GFAP and glutamate aspartate transporter (GLAST)-positive astrocytes. This decrease was reversed by the addition of CysC but not by another cysteine protease inhibitor. Thus, the promotion of astrocyte development by CysC appears to be independent of its protease inhibitor activity. The antibody increased the number of oligodendrocytes and their precursors. Therefore, CysC modifies glial development in addition to its activity against neural stem/precursor cells.

Age Factors↗

PubMed2006

Pup birth from mouse oocytes in preantral follicles derived from vitrified and warmed ovaries followed by in vitro growth, in vitro maturation, and in vitro fertilization.

OBJECTIVE: To determine an optimum condition for vitrification of various animal species and to examine the developing capacity into pups of mouse oocytes in preantral follicles originating from cryopreserved ovaries. DESIGN: Experimental animal study. SETTING: Laboratory environment. ANIMAL(S): Normal (C57BL/6 x DBA2) F1 mice. INTERVENTION(S): Vitrification of animal ovaries using polyester sheets as a storage device; collection of oocyte-granulosa cell complexes by enzymatic treatment; and in vitro growth (IVG), in vitro maturation (IVM), and IVF, with embryo transplantation to pseudopregnant mice. MAIN OUTCOME MEASURE(S): Histological analysis of vitrified and warmed ovaries from several animal species; measurement of successful rates in IVG, IVM, and IVF of mouse oocytes in oocyte-granulosa cell complexes collected from the vitrified and warmed ovaries; and achievement of pup birth. RESULT(S): The vitrification method used was effective for storage of various animal ovaries. The oocytes enclosed in preantral follicles that were yielded from the vitrified-warmed ovaries preserved the capacity for developing into pups after IVG, IVM, and IVF. CONCLUSION(S): The findings indicated that ovarian cryopreservation by vitrification and subsequent IVG, IVM, and IVF are promising methods for restoring young cancer patients' fertility. The practical information presented here is applicable to human ovarian tissues.

Animals↗

PubMed2006

Pig zona pellucida 2 (pZP2) protein does not participate in zona pellucida formation in transgenic mice.

The zona pellucida, an extracellular matrix surrounding mammalian oocytes, is composed of three or four glycoproteins. It is well known that the zona pellucida plays several critical roles during fertilization, but there is little knowledge about its formation. The purpose of this study is to examine whether a pig zona pellucida glycoprotein 2 (pZP2) would assemble with mouse zona pellucida. A transgene construct was prepared by placing a minigene encoding pZP2 downstream from the promoter of mouse ZP2. The result showed that the transgenic protein was synthesized in growing oocytes but not incorporated into the zona pellucida. Furthermore, the pZP2 transgene did not rescue the phenotype in ZP2-knockout zona-deficient mice. These results indicate that pZP2 does not participate in mouse zona pellucida formation and the zona pellucida is constituted from its component proteins in a molecular species-specific manner between mice and pigs.

Animals↗

PubMed2005

Immunogenicity and contraceptive potential of recombinant human sperm associated antigen (SPAG9).

Human sperm-associated antigen 9 (hSPAG9) is a potential target for sperm-based contraceptive vaccine in lieu of its location on the sperm acrosomal compartment and its implication in sperm-egg interaction. SPAG9 is an acrosomal molecule which is not only restricted to a specific region (domain) of the acrosome but also undergoes relocation to the equatorial region in a stage-specific manner during acrosome reaction, demonstrating its potential role in sperm-egg binding. Human SPAG9 nucleotide sequence revealed 94% identity with macaque SPAG9 and 96.8% with baboon SPAG9 over the entire sequence. The amino acid sequence comparison of human SPAG9 with macaque and baboon revealed an overall homology of 84.9% and 90.6%, respectively. The presence of a high level of homology at the amino acid and nucleotide levels indicates that SPAG9 is conserved in macaque, baboon and human sharing common function and common origin in the biological past. Immunogenicity studies were carried in rats, which demonstrated that recombinant hSPAG9 protein adsorbed on alum is highly immunogenic. Antibodies thus generated after immunization reacted with recombinant human SPAG9 (rhSPAG9) and native SPAG9 protein from human sperm in Western blot analysis. In an in vitro assay, anti-rhSPAG9 antibodies inhibited sperm adherence to or penetration in zona-free hamster egg penetration test. Further, anti-SPAG9 antibodies inhibited the binding of human sperm to intact human oocyte as well as to matched hemi-zonae, indicating that the recombinant protein is a suitable contraceptive vaccinogen. Together these results demonstrate that the rhSPAG9 adsorbed on alum is immunogenic in nature, which is a permissible adjuvant for immunogenicity and fertility trials in non-human primates.

Acrosome Reaction↗

PubMed2005

Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein.

We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561-565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an alpha-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38alpha or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa-egg interaction.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding.

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.

Amino Acid Sequence↗

PubMed2005

Target antigens for sperm-immobilizing antibodies found in infertile women.

Complement-dependent sperm-immobilizing antibodies (SI-Abs) have frequently been detected in the sera of infertile women. Analyses using monoclonal antibodies as well as patients' antisperm antibodies have shown that the carbohydrate moieties of sperm and seminal plasma are major epitopes of these antibodies. In the present study, we characterized the antigen molecules recognized by human monoclonal SI-Ab (MAb H6-3C4) derived from a patient with strong SI-Ab activity. Immunofluorescent staining showed that MAb H6-3C4 reacted with ejaculated human sperm and the epididymis but not with the testes and other somatic organs including the spleen. In Western blot analysis, MAb H6-3C4 reacted to antigen molecules of Mr 15-27 kDa present in the methanol-chloroform extracts of either ejaculated sperm or seminal plasma. Amino acid sequence analysis of the reactive molecule revealed that the amino acid sequence was identical to the core peptide of CD52, a GPI-anchored protein on lymphocytes. Furthermore, the epitope for MAb H6-3C4 was found to be the specific N-linked carbohydrate for the male reproductive tract. These results suggest that SI-Abs in some infertile women target a sperm-specific carbohydrate antigen on CD52 molecules which are secreted from the epididymis and coat the sperm surface.

Animals↗

PubMed2005

Follicular dysfunction induced by autoimmunity to zona pellucida.

The mammalian zona pellucida is an extracellular matrix that occurs in growing oocytes, ovulated eggs and pre-implantation embryos, and is known to be involved in several important events during ovarian folliculogenesis and fertilization. Since the zona pellucida is formed at an early stage of oocyte growth, circulating antibodies against zona pellucida may impair ovarian function. In this article we discuss whether anti-zona antibodies cause ovarian dysfunction and infertility. The discussion is based on clinical examination and animal experiments including the following approaches: 1/ immunological method using solubilized human zona pellucida detected anti-zona antibody with a high frequency in infertile patients, especially premature ovarian failure syndrome; 2/ in vivo experiment using hamsters showed that some, but not all, animals experienced ovarian failure after immunization with hamster recombinant zona proteins; 3/ in vitro experiment using mouse isolated ovarian follicles showed significant inhibitory effects on follicular growth and oocyte development. We concluded that anti-zona antibody may be involved in causing ovarian failure.

Animals↗

PubMed2004

In vitro growth and maturation as well as fertilization of mouse preantral oocytes from vitrified ovaries.

OBJECTIVE: To examine the capacity for fertilization and development of preantral oocytes in oocyte-granulosa cell complexes (OGC) originating from vitrified ovaries. DESIGN: Experimental animal study. SETTING: University-based research laboratory. ANIMAL(S): Normal (C57BL/6xDBA2) F1 mice in a laboratory environment. INTERVENTION(S): Vitrification of mouse ovaries using polyester sheets as a storage device; collection of OGC by enzymatic treatment; in vitro growth (IVG), in vitro maturation (IVM), and in vitro fertilization (IVF). MAIN OUTCOME MEASURE(S): We performed histologic analysis of vitrified and warmed ovaries, and measured the successful rate in IVG, IVM, and IVF of oocytes in the OGC collected from the ovaries. RESULT(S): The cortical region of ovaries maintained good morphologic structure after vitrification and warming. Upon IVG and IVM, 75.9% of oocytes in OGC matured to the metaphase II (MII) stage. The fertilization rate of these oocytes was 57.5% as compared with 69.5% for fresh ovaries. CONCLUSION(S): The vitrification method used was effective for storage of ovaries. The oocytes enclosed in preantral follicles from the ovaries preserved capacity for fertilization and development to preimplantation embryos.

Animals↗

PubMed2004

Birth of healthy neonates after intracytoplasmic injection of ejaculated or testicular spermatozoa from men with nonmosaic Klinefelter's syndrome: a report of 2 cases.

BACKGROUND: Klinefelter's syndrome is one of the major causes of azoospermia, cryptozoospermia and severe oligozoospermia with either a nonmosaic (47,XXY) or mosaic (47,XXY/46,XY) ICSI treatment with cryopreserved testicular spermatozoa failed, but after the third attempt, 6 of 8 oocytes injected with cryopreserved sperm were fertilized and karyotype. Men with Klinefelter's syndrome generally have difficulty having children. CASES: Patient 1 had motile spermatozoa in the ejaculate, which were injected into 3 oocytes, resulting in fertilization and cleavage. Two good-quality embryos were transferred into his wife's uterine cavity. She conceived and, following a normal pregnancy, delivered a healthy female infant. Two years later she conceived for the second time with motile spermatozoa in the ejaculate and delivered a healthy male infant uneventfully. To our knowledge, this was the first case in which a nonmosaic Kleinefelter's syndrome patient fathered 2 children through intracytoplasmic sperm injection (ICSI) using motile spermatozoa in the ejaculate. Patient 2, with azoospermia, was subjected to testicular biopsy to collect spermatozoa. The first 2 attempts at ICSI treatment with cryopreserved testicular spermatozoa failed, but after the third attempt, 6 of 8 oocytes injected with cryopreserved sperm were fertilized and cleaved. Two of these embryos were transferred into the wife's uterine cavity. She conceived and, following a normal pregnancy, delivered a healthy male infant. In all cases, amniocentesis followed by genetic analysis showed a normal karyotype. CONCLUSION: Two infertile men with nonmosaic Klinefelter's syndrome successfully fathered normal children after intracytoplasmic injection of ejaculated or testicular spermatozoa.

Adult↗

PubMed2003

Efficacy of antibodies against Escherichia coli expressed chimeric recombinant protein encompassing multiple epitopes of zona pellucida glycoproteins to inhibit in vitro human sperm-egg binding.

To minimize ovarian dysfunction subsequent to immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing the antigenic B cell epitopes as immunogens have been proposed. In this study, attempts have been made to clone and express a recombinant chimeric protein encompassing the epitopes corresponding to bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1, amino acid residues 132-147), ZP glycoprotein-2 (bmZP2, amino acid residues 86-113), and ZP glycoprotein-3 (bmZP3, amino acid residues 324-347). The above chimeric recombinant protein (r-bmZP123) was expressed as a polyhistidine fusion protein in Escherichia coli. Immunoblot with murine monoclonal antibody, MA-813, generated against recombinant bmZP1 revealed a major band of approximately 10 kDa. The r-bmZP123 was purified on nickel-nitrilotriacetic acid resin under denaturing conditions. The female rabbits immunized with purified r-bmZP123 conjugated to diphtheria toxoid (DT) generated antibodies that reacted with r-bmZP123 and DT in an ELISA. In addition, the immune sera also reacted with E. coli expressed recombinant bmZP1, bmZP2, and bmZP3. In an indirect immunofluorescence assay, the antibodies against r-bmZP123 recognized native ZP of bonnet monkey as well as human. The immune sera also inhibited, in vitro, the binding of human spermatozoa to the human zona in the hemizona assay (HZA). These studies, for the first time, demonstrate the feasibility of assembling multiple epitopes of different ZP glycoproteins as a recombinant protein that elicit antibodies which are reactive with native zona and also inhibit, in vitro, human sperm-oocyte binding.

Amino Acid Sequence↗

PubMed2003

A digital method of sperm immobilization test: comparison to the conventional method.

Antisperm antibodies have been found in infertile patients and those causing immobilization of sperm are considered to be closely related to unexplained infertility. These antibodies are usually identified by a sperm immobilization test which involves counting motile sperm under microscope. This test is subjective as it relies on the judgement of the examiner with respect to sperm motility. In this study, we analyzed motile sperm by a digital method using Sperm Quality Analyzer. The results were compared with those obtained by the conventional method. We found that the two methods yielded identical results, with 14 of 66 samples tested being positive and 52 negative for sperm immobilizing antibodies. These results show that the digital method is objective and of value in the measurement of motile sperm in determination of sperm immobilizing antibodies.

Agglutination Tests↗

PubMed2002

Antibodies generated in response to plasmid DNA encoding zona pellucida glycoprotein-B inhibit in vitro human sperm-egg binding.

To investigate the immunogenicity of plasmid DNA encoding bonnet monkey (Macaca radiata) zona pellucida (ZP) glycoprotein-B (bmZPB), the cDNA corresponding to bmZPB, excluding the N-terminal signal sequence and C-terminus transmembrane-like domain, was cloned in mammalian expression vector VR1020 downstream of tissue plasminogen activator signal sequence under cytomegalovirus promoter (VRbmZPB). In vitro transfection of COS-1, COS-7, CHO, HEK-293, and UM-449 mammalian cells with VRbmZPB plasmid DNA led to the expression of bmZPB. Expression of bmZPB in transfected cells was cytosolic. Flow cytometry analysis of COS-1 cells transfected with VRbmZPB revealed that approximately 15% cells expressed bmZPB. The expressed bmZPB has an apparent molecular weight of 57 kDa. Immunization of male BALB/cJ mice with VRbmZPB plasmid DNA in saline as compared to VR1020 immunized group, elicited significant antibodies against E. coli expressed recombinant bmZPB as evaluated in ELISA. The antibodies generated by VRbmZPB plasmid DNA recognized bonnet monkey as well as human ZP. The immune sera obtained from mice immunized with VRbmZPB plasmid DNA also inhibited, in vitro, the binding of spermatozoa to the ZP in the hemizona assay. These studies, for the first time, demonstrate the feasibility of DNA vaccine to generate antibodies against ZP that recognize native protein and inhibit human sperm-oocyte binding.

Animals↗

PubMed2002

Contraceptive potential of synthetic peptides of zona pellucida protein (ZPA).

Previously, we produced a fertilization-blocking monoclonal antibody (MAb-5H4) to find a candidate peptide for a contraceptive vaccine. MAb-5H4 recognized a linear amino acid sequence of ZPA (No. 50-67) in pigs, humans and rabbits. In the present study, 18mer peptides corresponding to the sequence were conjugated with diphtheria toxoid as a carrier protein before immunization in rabbits. All three antisera recognized human zona pellucida on testing by immunofluorescent staining method. The two produced against human and rabbit peptides effectively inhibited human sperm binding to the zona pellucida, but the antiserum against the pig peptide did not. The former two peptides include an identical sequence (LDPEKLTL) of the minimum binding motif for MAb-5H4, but the latter peptide includes one amino acid replacement (K to N) in the sequence. It is thus concluded that a synthetic peptide including the sequence of LDPEKLTL could be a feasible candidate for developing a contraceptive vaccine for humans.

Amino Acid Sequence↗

PubMed2002

Prospect for immunocontraception using the NH2-terminal recombinant peptide of human zona pellucida protein (hZPA).

PROBLEM: The zona pellucida is a feasible candidate for contraceptive vaccine. Our previous study showed that a human zona pellucida (ZPA) recombinant protein produced antiserum that inhibited human in vitro fertilization in rabbits. The objective of this study is to examine whether the human recombinant protein produce fertilization-blocking antiserum in a non-human primate. METHOD OF STUDY: The amino terminal regions consisting of pig (198 amino acids) and human (206 amino acids) zona pellucida proteins were prepared by Escherichia coli to produce antisera in bonnet monkeys (Macaca radiata). Antibody production was assessed by immunofluorescent staining and enzyme-linked immunosorbent assay (ELISA). Fertilization-blocking test was carried out using human oocytes that had failed in intracytoplasmic sperm injection (ICSI) treatment under patient's informed consent. RESULTS: Both antisera against pig and human recombinant proteins recognized native human zona pellucida by immunofluorescent staining. However, anti-recombinant human ZPA reacted to native pig zona protein more strongly than pig zona pellucida by ELISA. When human oocytes were treated with antisera before insemination, anti-human ZPA antiserum inhibited human sperm binding to human zona pellucida, but anti-pig ZPA antiserum did not. CONCLUSIONS: Bonnet monkey produced fertilization-blocking antibody by immunization with human recombinant ZPA. The homologous or highly similar sequence is effective for developing a contraceptive vaccine when using recombinant peptides.

Animals↗

PubMed2002

Nasal immunization with diphtheria toxoid conjugated-CD52 core peptide induced specific antibody production in genital tract of female mice.

PROBLEM: The common mucosal immune system (CMIS) has developed as a barrier for the numerous encounters between the host and various pathogens. It is possible to exploit this system to induce secretion of IgA antibody which inhibits sperm penetration in the female genital tract. In this study, the immunogenicity of a human sperm surface antigen (CD52) introduced by intranasal immunization was investigated with a view to developing a contraceptive vaccine. METHOD OF STUDY: A synthetic peptide corresponding to CD52 core peptide (GQNDTSQTSSPS) was prepared and conjugated with diphtheria toxoid (DT) as a carrier protein. The immunogen was given to mice with DOTAP:cholesterol liposome adjuvant intranasally, followed by determination of Ig and IgA class antibody levels in the sera and vaginal washes. RESULTS: The CD52 core peptide elicited IgA class as well as Ig antibodies both in the sera and vaginal secretions after intranasal immunization. An additional nasal inoculation after decrease of the antibody titer raised the antibody level to its highest level during the experiment. CONCLUSIONS: These results indicate that the CMIS could induce Ig and IgA class antibodies reactive to CD52 core peptide in the female genital tract. Intranasal immunization of a sperm-specific antigen would be a promising regimen for a safe and easy contraceptive vaccine.

Animals↗

PubMed2002

Old Yellow Enzyme from Candida macedoniensis catalyzes the stereospecific reduction of the C=C bond of ketoisophorone.

Microorganisms were screened for ones that reduced 3,5,5-trimethyl-2-cyclohexene-1,4-dione (ketoisophorone; KIP), and several strains were found to produce (6R)-2,2,6-trimethylcyclohexane-1,4-dione (levodione). The enzyme catalyzing the reduction of the C=C bond of KIP to yield (6R)-levodione was isolated from Candida macedoniensis AKU4588. The results of primary structural analysis and its enzymatic properties suggested that the enzyme might be an Old Yellow Enzyme family protein.

Amino Acid Sequence↗

PubMedDate not supplied

Native zona pellucida reactivity and in-vitro effect on human sperm-egg binding with antisera against bonnet monkey ZP1 and ZP3 synthetic peptides.

In mammals, zona pellucida glycoprotein-3 (ZP3) is the putative ligand for primary sperm binding and induces the acrosome reaction. Recent evidence suggests that zona pellucida glycoprotein-1 (ZP1) also play an important role, in some species, during fertilization. In order to identify synthetic peptide immunogens capable of inducing antibodies reactive with native zona and inhibiting sperm-oocyte interaction, peptide encompassing the amino acid (aa) residues 334-343 of bonnet monkey ZP3 (bmZP3) was synthesized co-linearly with a 'promiscuous' T-cell epitope of circumsporozoite protein (CSP, 378-398 aa) of Plasmodium falciparum. In addition, four peptides corresponding to bonnet monkey ZP1 (bmZP1((58-79 aa)), bmZP1((136-153 aa)), bmZP1((212-228 aa)) and bmZP1((251-273 aa))) were synthesized. The synthetic peptides corresponding to bmZP1 were conjugated with diphtheria toxoid. Immunization of female BALB/cJ mice with the above conjugates and CSP-bmZP3((334-343 aa)) peptide led to the generation of an adequate antibody response against the respective zona peptide. Antibodies against bmZP1((251-273 aa)) and CSP-bmZP3((334-343 aa)) recognized bonnet monkey and human zona pellucida in an indirect immunofluorescence assay. Further, these antibodies when tested independently or in combination also significantly inhibited the binding of human spermatozoa to zona pellucida in a hemizona assay. These studies will further help in the design of synthetic peptide immunogens comprising of multiple B cell epitope from different zona proteins for better immunocontraceptive efficacy.

Amino Acid Sequence↗