• Asian-Australasian Journal of Animal Sciences
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• v.21 no.5
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• pp.754-768
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• 2008

The past decades have seen enormous advances in our understanding of how GH acts. GH is a pituitary-derived polypeptide hormone that has diverse physiological effects including the regulation of bone growth, carbohydrate and lipid metabolism. The effects of GH are mediated directly and indirectly through IGF-I. In addition, GH stimulates the hepatic production of ALS. In postnatal life, IGF-I and -II circulate as 150 kDa ternary complexes consisting of one molecule each of IGFBP-3 or IGFBP-5, IGF-I or IGF-II and ALS. It is now known that ALS increases significantly the half-lives of the IGFs, IGFBP-3 and -5, and therefore is responsible for maintaining a circulating reservoir for each of these proteins.

• Journal of Animal Science and Technology
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• v.46 no.4
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• pp.555-562
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• 2004

본 연구는 목저은 다음과 같다: 1) 돼지에서 150-kDa temary insulin-like growth faetor(IGF)complex의 한 구성 요소인 acid-labile subunit(ALS) 유전자 intron의 존재 확인. cloning 및 돼지 ALS(porcine ALS; pALS) complementary DNA(cDNA)의 3' 비해독(untranslated) 부위(3' UT) 증폭. cloning, 2) intron-spanning primer pair를 이용한 reverse transcription-polymerase chain reaction(RT-PCR) 방법에 의한 돼지 조직에서의 ALS 유전자 발현 분포 확인 및 3) 돼지 hepatocyte에서의 ALS 유전자 발현 여부 확인. 돼지 genomic DNA를 template로 하여 PCR 방법으로 예상된는 intron 부위를 증폭하고 plasmid vector에 삽입하여 염기서열을 결정한 결과 타 종의 ALS 유전자에서와 같은 위치에 1,371-base pair(bp)의 pALS intron이 존재함을 확인하였다. 역시 본 연구에서 간에서 추출한 RNA를 주형으로 시작하여 3' rapid amplification of cDNA end(3' RACE) 방법으로 147-bp의 3'UT를 합성하고 그 염기성열을 결정하였다. RT-PCR 결과 간은 물론 조사된 모든 돼지의 내장기관(신장, 폐, 비장)과 자성 생식기관(난소, 난관, 자궁) 및 골격근육에서 ALS 유전자가 발현됨이 밝혀졌다. 또한 돼지 간 조직에 대한 in-situ hybridization 결과 hepatocyte에서 ALS 유전자가 발현됨이 확인되었다. 이상의 결과는 ALS가 혈중 IGF의 저정/조절체로서의 주기능 외에 모세혈관 밖에서도 미지의 기능이 있을 기능성을 시사한다.

• BMB Reports
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• v.37 no.2
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• pp.153-158
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• 2004

The acid-labile subunit (ALS) is known to interact with the IGF binding protein (IGFBP) in the presence of insulin-like growth factors (IGFs). Studies, however, indicate that ALS forms a doublet with IGFBP3, independent of IGFs. To characterize the structural domain required for the IGF-free ALS-IGFBP3 interaction, seven recombinant human IGFBP3 mutants were generated: three deletion mutants and four site-specific mutants that had altering N-terminal regions of IGFBP3. ALS and IGFBP3 mRNAs were co-injected into Xenopus oocytes, and their products were cross-linked and immunoprecipitated using antisera against ALS or IGFBP3. Among the deletion mutants, the mutant of D40 (deleted in 11-40th amino acids) exerted no effect in the interaction with ALS, while D60 (${\Delta}11$-60) demonstrated a moderate reduction. D88 (${\Delta}11$-88), however, showed a significant decrease. In the case of site-specific mutants, the mutation that alterated the IGF binding site (codons 56 or 80) exerted a significant reduction in the interaction, whereas codons 72 or 87 showed no significant change in the interaction with ALS. The stability of the ALS-IGFBP3 interaction was analyzed according to a time-dependent mode. Consistent with the binding study, mutants on the IGF binding sites (56 or 80) consistently show a weakness in the ALS-IGFBP3 interaction when compared to the mutants that covered the non-IGF binding sites (72 or 87). This study suggests that the N-terminal of IGFBP3, especially the IGF binding site, plays an important role in interacting with ALS as well as in stabilizing the dual complex, independent of IGFs.

• Journal of Animal Science and Technology
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• v.50 no.2
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• pp.177-184
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• 2008

Acid-labile subunit(ALS) is a 85-kDa glycosylated plasma protein which forms a 150-kDa ternary complex with 7.5-kDa insulin-like growth factor(IGF) and 40~45-kDa IGF-binding protein-3. In a previous study, the present authors prepared a porcine ALS(pALS) expression construct by inserting a pALS coding sequence into a plasmid vector following synthesis of the sequence by reverse transcription-polymerase chain reaction(RT-PCR). The expression construct, however, was subsequently found to have a mis-sense mutation at two bases of the pALS coding sequence which is presumed to have occurred through a PCR error. In the present study, the correct coding sequence was synthesized by the site-directed mutagenesis and inserted into the pET-28a(+) plasmid expression vector containing the His-tag sequence flanking the last codon of the insert DNA. After induction of the expression construct in E. coli BL21(DE3) cells, the resulting presumptive recombinant peptide was purified by the Ni-affinity chromatography. Upon SDS- PAGE, the affinity-purified peptide was resolved as a single band at a 66-kDa position which is consistent with the expected molecular mass of the presumptive recombinant pALS. Collectively, results indicate that a recombinant pALS peptide was successfully expressed and purified in the present study.

• BMB Reports
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• v.35 no.2
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• pp.186-193
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• 2002

The acid-bible subunit (ALS) associates with the insulinlike growth factor (IGF)-I or II, and the IGF binding protein-3 (IGFBP-3) in order to form a 150-kD complex in the circulation. This complex may regulate the serum IGFs by restricting them in the vascular system and promoting their endocrine actions. Little is known about how ALS binds to IGFBP3, which connects the IGFs to ALS. Xenopus oocyte was utilized to study the function of ALS in assembling IGFs into the ternary complexes. Xenopus oocyte was shown to correctly translate in vitro transcribed mRNAs of ALS and IGFBP3. IGFBP3 and ALS mRNAs were injected in a mixture, and their products were immunoprecipitated by antisera against ALS and IGFBP3. Contrary to traditional reports that ALS interacts only with IGF-bound IGFBP3, this study shows that ALS is capable of forming a binary complex with IGFBP3 in the absence of IGF When cross-linked by disuccinimidyl suberate, the band that represents the ALS-IGFBP3 complex was evident on the PAGE. IGFBP3 movement was monitored according to the distribution between the hemispheres. Following a localized translation in the vegetal hemisphere, IGFBP3 remained in the vegetal half in the presence of ALS. However, the mutant IGFBP3 freely diffused into the animal half, despite the presence of ALS, which is different from the wild type IGFBP3. This study, therefore, suggests that ALS may play an important role in sequestering IGFBP3 polypeptides via the intermolecular aggregation. Studies using this heterologous model will lead to a better understanding of the IGFBP3 and ALS that assemble into the ternary structure and circulate the IGF system.

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.226.2-226.2
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• 2003

The FimH subunit of type 1-fimbriated Escherichia coli has been determined as a major cause of urinary tract infection. To produce a possible vaccine antigen against urinary tract infection, the fimH gene was genetically linked to the Itxa2b gene, which was then cloned into the pMAL -p2E expression vector. The chimaeric construction of pMALfimH/Itxa2b was transformed into Escherichia coli TB1 and its N-terminal amino acid sequence was analyzed. (omitted)

• BMB Reports
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• v.38 no.2
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• pp.225-231
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• 2005

Acid-labile subunit (ALS) is a component of the 150-kDa insulin-like growth factor-binding protein-3 (IGFBP-3) complex, which, by sequestering the majority of IGFs-I and -II and thereby prolonging the half-life of them in plasma, serves as a circulating reservoir of IGFs in mammalian species. A pGEX-2T plasmid and a baculovirus expression constructs harboring a coding sequence for glutathione-S transferase (GST)-porcine ALS (pALS) fusion protein were expressed in BL21(DE3) E. coli and Sf9 insect cells, respectively. The expressed protein was purified by glutathione or Ni-NTN affinity chromatography, followed by cleavage of the fusion protein using Factor Xa. In addition, pALS and hIGFBP-3 were also produced in small amounts in the Xenopus oocyte expression system which does not require any purification procedure. A 65-kDa pALS polypeptide was obtained following the prokaryotic expression and the enzymatic digestion, but biochemical characterization of this polypeptide was precluded because of an extremely low expression efficiency. The baculovirus-as well as Xenopus-expressed pALS exhibited the expected molecular mass of 85 kDa which was reduced into 75 and 65 kDa following deglycosylation of Asn-linked carbohydrates by Endo-F glycosidase, indicating that the expressed pALS was properly glycosylated. Moreover, irrespective of the source of pALS, the recombinant pALS and hIGFBP-3 formed a 130-kDa binary complex which could be immunoprecipitated by anti-hIGFBP-3 antibodies. Collectively, results indicate that an authentic pALS protein can be produced by the current expression systems.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2001.11a
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• pp.99-99
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• 2001

The acid-labile subunit (ALS) associates with insulin-like growth factor (IGF)-I or -II and IGF binding protein-3 (IGFBP-3) to form a 150-kD complex in the circulation. This complex is thought to regulate the serum IGFs by restricting them in the vascular system and promotes their endocrine actions. Little is known about how ALS binds to IGFBP3, which connects the IGFs to ALS. Xenopus oocyte was utilized to study the function of ALS in assembling IGFs into the ternary complexes. Xenopus oocyte was shown to correctly translate in vitro transcribed mRNAs of ALS and IGFBP3. IGFBP3 and ALS mRNAs were injected in mixture and their products were immunoprecipitated by antisera against ALS and IGFBP3. Contrary to the traditional reports that ALS interacts only with IGF-bound IGFBP3, this study shows that ALS is capable of forming a binary complex with IGFBP3 in the absence of IGF. When cross-linked by disuccinimidyl substrate, band representing ALS-IGFBP3 complex was evident on the PAGE. IGFBP3 movement was monitored according to the distribution between the hemispheres. Following a localized translation in the vegetal hemisphere, IGFBP3 was shown to remain in the vegetal half in the presence of ALS. Different from wild type IGFBP3, however, mutant IGFBP3 freely diffused into the animal half despite the presence of ALS. Taken together, this study suggests that ALS may play an important role in sequestering IGFBP3 polypeptides via the intermolecular aggregation. Studies using this heterologous model will lead to a better understanding of the IGFBP3 and ALS assembling into the ternary structure and circulating IGF system.

• Biomolecules & Therapeutics
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• v.13 no.2
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• pp.101-106
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• 2005

The generation of secretory IgA antibodies (Abs) for specific immune protection of mucosal surfaces depends on stimulation of the mucosal immune system, but this is not effectively achieved by parenteral or even oral administration of most soluble antigens. Thus, to produce a possible vaccine antigen against urinary tract infections, the uropathogenic E. coli (UPEC) adhesin was genetically coupled to the heat-labile Escherichia coli enterotoxin A2B (ltxa2b) gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimH/ltxa2b was then transformed into E. coli K-12 TB1 and its nucleotide sequence was verified. The chimeric protein was then purified by applying the affinity chromatography. The purified chimeric protein was confirmed by SDS-PAGE and westem blotting using antibodies to the maltose binding protein (MBP) or the heat labile E. coli subunit B (LTXB), plus the N-terminal amino acid sequence was analyzedd. The orderly-assembled chimeric protein was confirmed by a modified $G_{M1}$-ganglioside ELISA using antibodies to adhesin. The results indicate that the purified chimeric protein was an Adhesin/LTXA2B protein containing UPEC adhesin and the $G_{M1}$-ganglioside binding activity of LTXB. thisstudy also demonstrate that peroral administration of this chimeric immunogen in mice elicited high level of secretory IgA (sIgA) and serum IgG Abs to the UPEC adhesin. The results suggest that the genetically linked LTXA2B acts as a useful mucosal adjuvant, and that adhesin/LTXA2A chimeric protein might be a potential antigen for oral immunization against UPEC.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.12
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• pp.1668-1676
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• 2010

The somatotropic axis plays a key role in proliferation and differentiation of avian organs during both pre- and posthatching periods. This review discusses the complexity of regulation of the endocrine system for chicken development and growth by growth hormone (GH), insulin-like growth factor (IGF), and IGF binding protein (IGFBP). In addition, the thyrotropic axis, including thyrotropin-releasing hormone (TRH) and thyroid hormones ($T_4$ and $T_3$), is also involved in the GH-secreting pattern. In mammals, IGFI and -II are always sequestered in a 150 kDa non-covalent ternary complex. This complex consists of one molecule each of IGF-I or IGF-II, IGFBP-3 or IGFBP-5 and an acid labile subunit (ALS). Chick ALS is identified in different strains for the first time, and further investigation of the expression of ALS on developmental stage and ALS effect on IGF bioavailability may be addressed in the future.