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You-Min Feng

Publications and source records attributed to You-Min Feng.

At least 19 recordsLinked to original sources
PubMed2003

Refolding of amphioxus insulin-like peptide: implications of a bifurcating evolution of the different folding behavior of insulin and insulin-like growth factor 1.

Insulin and insulin-like growth factor 1 (IGF-1) share high sequence homology, but their folding behaviors are significantly different: insulin folds into one unique thermodynamically controlled structure, while IGF-1 folds into two thermodynamically controlled disulfide isomers. However, the origin of their different folding behaviors is still elusive. The amphioxus insulin-like peptide (ILP) is thought to be the common ancestor of insulin and IGF-1. A recombinant single-chain ILP has been expressed previously, and now its folding behavior is investigated. The folding behavior of ILP shows the characteristics of both insulin and IGF-1. On one hand, two thermodynamically controlled disulfide isomers of ILP have been identified; on the other hand, the content of isomer 1 (its disulfides are deduced identical to those of swap IGF-1) is much less than that of isomer 2 (its disulfides are deduced identical to those of native IGF-1); that is, more than 96% of ILP folds into the native structure. The present results suggest that the different folding behaviors of insulin and IGF-1 are acquired through a bifurcating evolution: the tendency of forming the thermodynamically controlled non-native disulfide isomer is diminished during evolution from ILP to insulin, while this tendency is amplified during evolution from ILP to IGF-1. Moreover, the N-terminal Gln residue of ILP can spontaneously form a pyroglutamate residue, and its cyclization has a significant effect on the folding behavior of ILP: the percentage of isomer 1 is approximately 2-fold that of isomer 1 of the noncyclized ILP; that is, isomer 1 becomes more favored when the N-terminal residue of ILP is cyclized. So, we deduce that the N-terminal residues have a significant effect on the folding properties of insulin, IGF-1, and ILP.

Amino Acid Sequence↗
PubMed2003

In vitro refolding of human proinsulin. Kinetic intermediates, putative disulfide-forming pathway folding initiation site, and potential role of C-peptide in folding process.

Human insulin is a double-chain peptide that is synthesized in vivo as a single-chain human proinsulin (HPI). We have investigated the disulfide-forming pathway of a single-chain porcine insulin precursor (PIP). Here we further studied the folding pathway of HPI in vitro. While the oxidized refolding process of HPI was quenched, four obvious intermediates (namely P1, P2, P3, and P4, respectively) with three disulfide bridges were isolated and characterized. Contrary to the folding pathway of PIP, no intermediates with one- or two-disulfide bonds could be captured under different refolding conditions. CD analysis showed that P1, P2, and P3 retained partially structural conformations, whereas P4 contained little secondary structure. Based on the time-dependent distribution, disulfide pair analysis, and disulfide-reshuffling process of the intermediates, we have proposed that the folding pathway of HPI is significantly different from that of PIP. These differences reveal that the C-peptide not only facilitates the folding of HPI but also governs its kinetic folding pathway of HPI. Detailed analysis of the molecular folding process reveals that there are some similar folding mechanisms between PIP and HPI. These similarities imply that the initiation site for the folding of PIP/HPI may reside in the central alpha-helix of the B-chain. The formation of disulfide A20-B19 may guide the transfer of the folding information from the B-chain template to the unstructured A-chain. Furthermore, the implications of this in vitro refolding study on the in vivo folding process of HPI have been discussed.

Amino Acid Sequence↗
PubMed2003

A peptide model of insulin folding intermediate with one disulfide.

Insulin folds into a unique three-dimensional structure stabilized by three disulfide bonds. Our previous work suggested that during in vitro refolding of a recombinant single-chain insulin (PIP) there exists a critical folding intermediate containing the single disulfide A20-B19. However, the intermediate cannot be trapped during refolding because once this disulfide is formed, the remaining folding process is very quick. To circumvent this difficulty, a model peptide ([A20-B19]PIP) containing the single disulfide A20-B19 was prepared by protein engineering. The model peptide can be secreted from transformed yeast cells, but its secretion yield decreases 2-3 magnitudes compared with that of the wild-type PIP. The physicochemical property analysis suggested that the model peptide adopts a partially folded conformation. In vitro, the fully reduced model peptide can quickly and efficiently form the disulfide A20-B19, which suggested that formation of the disulfide A20-B19 is kinetically preferred. In redox buffer, the model peptide is reduced gradually as the reduction potential is increased, while the disulfides of the wild-type PIP are reduced in a cooperative manner. By analysis of the model peptide, it is possible to deduce the properties of the critical folding intermediate with the single disulfide A20-B19.

Animals↗
PubMed2003

Peptide models of four possible insulin folding intermediates with two disulfides.

The single-chain insulin (PIP) can spontaneously fold into native structure through preferred kinetic intermediates. During refolding, pairing of the first disulfide A20-B19 is highly specific, whereas pairing of the second disulfide is likely random because two two-disulfide intermediates have been trapped. To get more details of pairing property of the second disulfide, four model peptides of possible folding intermediates with two disulfides were prepared by protein engineering, and their properties were analyzed. The four model peptides were named [A20-B19, A7-B7]PIP, [A20-B19, A6-B7]PIP, [A20-B19, A6-A11]PIP, and [A20-B19, A7-A11]PIP according to their remaining disulfides. The four model peptides all adopt partially folded structure with moderate conformational differences. In redox buffer, the disulfides of the model peptides are more easily reduced than those of the wild-type PIP. During in vitro refolding, the reduced model peptides share similar relative folding rates but different folding yields: The refolding efficiency of the reduced [A20-B19, A7-A11]PIP is about threefold lower than that of the other three peptides. The present results indicate that the folding intermediates corresponding to the present model peptides all adopt partially folded conformation, and can be formed during PIP refolding, but the chance of forming the intermediate with disulfide [A20-B19, A7-A11] is much lower than that of forming the other three intermediates.

Chromatography, High Pressure Liquid↗
PubMed2003

Contribution of the absolutely conserved B8Gly to the foldability of insulin.

B8Gly is absolutely conserved in insulin from different species, and in other members of the insulin superfamily the corresponding position is always occupied by a Gly residue. However, the reasons for its conservation are still unclear; probably many factors contribute to this phenomenon. In our previous work, B8Gly was replaced by an Ala residue, which suggested that biological activity is one of the factors contributing to its conservation. In order to identify more factors contributing to this positional conservation, the secretion efficiency, structural stability, disulfide stability, and in vitro refolding of single-chain insulin (PIP) and a mutant with B8Gly replaced by Ala, were investigated. Compared with wild-type PIP, the B8Ala replacement decreased the secretion efficiency, structural stability, disulfide stability, and in vitro refolding efficiency of the PIP sequence. These results suggest that B8Gly is important to the secretion, folding, and stability of the insulin sequence.

Alanine↗
PubMed2002

The different energetic state of the intra A-chain/domain disulfide of insulin and insulin-like growth factor 1 is mainly controlled by their B-chain/domain.

Insulin and insulin-like growth factor 1 (IGF-1) share homologous sequence, similar three-dimensional structure, and weakly overlapping biological activity, but different folding information is stored in their homologous sequences: the sequence of insulin encodes one unique thermodynamically stable three-dimensional structure while that of IGF-1 encodes two disulfide isomers with different three-dimensional structure but similar thermodynamic stability. Their different folding behavior probably resulted from the different energetic state of the intra A-chain/domain disulfide: the intra A-chain disulfide of insulin is a stable bond while that of IGF-1 is a strained bond with high energy. To find out the sequence determinant of the different energetic state of their intra A-chain/domain disulfide, the following experiments were carried out. First, a local chimeric single-chain insulin (PIP) with the A8-A10 residues replaced by the corresponding residues of IGF-1 was prepared. Second, the disulfide stability of two global hybrids of insulin and IGF-1, Ins(A)/IGF-1(B) and Ins(B)/IGF-1(A), was investigated. The local segment swap had no effect on the fidelity of disulfide pairing and the disulfide stability of PIP molecule although the swapped segment is close to the intra A-chain/domain disulfide. In redox buffer which favors the disulfide formation for most proteins, Ins(A)/IGF-1(B) cannot form and maintain its native disulfides just like that of IGF-1, while the disulfides of Ins(B)/IGF-1(A) are stable in the same condition. One major equilibrium intermediate with two disulfides of Ins(A)/IGF-1(B) was purified and characterized. V8 endoproteinase cleavage and circular dichroism analysis suggested that the intra A-chain/domain disulfide was reduced in the intermediate. Our present results suggested that the energetic state of the intra A-chain/domain disulfide of insulin and IGF-1 was not controlled by the A-chain/domain sequence close to this disulfide but was mainly controlled by the sequence of the B-chain/domain.

Chromatography, High Pressure Liquid↗
PubMed2002

In vitro evolution of amphioxus insulin-like peptide to mammalian insulin.

By site-directed mutagenesis, six insulin residues related to the insulin-receptor interaction were grafted, partially or fully, onto the corresponding position of a recombinant amphioxus insulin-like peptide (ILP) that contained the A- and B-domains of the deduced amphioxus ILP. After fermentation, purification, and enzymatic cleavage, six insulin-like double-chain ILP analogues were obtained: [A2Ile]ILP, [B12Val, B16Tyr]ILP, [B25Phe]ILP, [A2Ile, B12Val, B16Tyr, B25Phe]ILP (four-mutated ILP), [A2Ile, B12Val, B16Tyr, B24Phe, B25Phe]ILP (five-mutated ILP), and [A2Ile, B12Val, B16Tyr, B24Phe, B25Phe, B26Tyr]ILP (six-mutated ILP). Circular dichroism analysis showed that such replacement did not significantly affect their secondary and tertiary structure compared with that of the wild-type ILP. The insulin-receptor-binding activity of the four-, five-, and six-mutated ILP was 0.14%, 11%, and 11% of native insulin, respectively; the other three ILP analogues acquired none of the detectable insulin-receptor-binding potency. The growth-promoting activities of the five- and six-mutated ILP were both about 50% of native insulin, while that of the wild-type ILP was not detectable. By structure-function-based mutagenesis, the completely inactive amphioxus ILP was converted into a molecule with moderate mammalian insulin activity. These results indicated the following: first, the grafted as well as those inborn insulin-receptor-binding related residues can form an insulin-receptor-binding patch on the ILP analogues; second, the ILP can be used as a scaffold molecule to investigate the role of the insulin residues; third, the natural evolution of amphioxus ILP to mammalian insulin is a possible process and can be mimicked in the laboratory.

Amino Acid Sequence↗
PubMed2002

The different folding behavior of insulin and insulin-like growth factor 1 is mainly controlled by their B-chain/domain.

Although insulin and insulin-like growth factor 1 (IGF-1) share homologous sequence, similar tertiary structure, weakly overlapped biological activity, and a common ancestor, the two highly homologous sequences encode different folding behavior: insulin folds into one unique stable tertiary structure while IGF-1 folds into two disulfide isomers with similar thermodynamic stability. To further elucidate the molecular mechanism of their different folding behavior, we prepared two single-chain hybrids of insulin and IGF-1, Ins(A)/IGF-1(B) and Ins(B)/IGF-1(A), as well as a mini-IGF-1 by means of protein engineering and studied their structure as well as folding behavior. Both mini-IGF-1 and Ins(A)/IGF-1(B) fold into two thermodynamically stable disulfide isomers in vivo and in vitro just like that of IGF-1, while Ins(B)/IGF-1(A) folds into one unique thermodynamically stable tertiary structure in vivo and in vitro just like that of insulin. So we deduce that the different folding behavior of insulin and IGF-1 is mainly controlled by their B-chain/domain. By V8 endoproteinase digestion and circular dichroism analysis, as well as insulin receptor binding assay, we deduce that Ins(B)/IGF-1(A), isomer 2 of mini-IGF-1, and isomer 2 of Ins(A)/IGF-1(B) adopt native IGF-1/insulin-like three-dimensional structure with native disulfides, while isomer 1 of mini-IGF-1 and isomer 1 of Ins(A)/IGF-1(B) adopt the swap IGF-1-like three-dimensional structure with swap disulfides.

Amino Acid Sequence↗
PubMed2002

[Preparation, characterization and receptor-binding capacity of pig serum transferrin half-molecules containing a single iron-binding site].

N- and C-half molecules containing a single iron-binding site were simultaneously obtained from trypsin digest of iron-saturated pig transferrin. The activities of the pig serum transferrin and of its N- and C-half molecules to bind the human placental membrane transferrin receptor were compared. The results indicate that the receptor-binding site of pig transferrin may be located at the C-half molecule of the transferrin.

Amino Acids↗
PubMed2001

Unfolding of Recombinant Single-chain Insulin in Denaturants Containing Thiol Reagents.

Recombinant single-chain insulin (PIP) contains three disulfide bonds. In the presence of denaturants and thiol reagents, the native structure of PIP was disturbed and its native disulfides were shuffled to form a mixture of scrambled isomers which have different degrees of unfolding. In this paper the unfolding degrees of PIP in urea or guanidine hydrochloride containing 0.2 mmol/L 2-mercaptoethanol was analyzed by reverse-phase HPLC and far-UV circular dichroism(CD). The peptide mapping of PIP scrambles demonstrated that PIP had shuffled its native disulfides under the condition we used. Among others, a major non-natural PIP disulfide isomer was purified and its refolding in vitro was investigated. These results show that PIP has only one thermodynamically stable disulfide linkage, and the non-natural disulfide isomers could refold in vitro efficiently to from native PIP. On basis of these, the differences between PIP, IGF-I and insulin on unfolding and refolding were discussed.

Journal Article↗
PubMed2000

Relationship between insulin A chain regions and insulin biological activities.

AIM:To study the relationship between insulin A chain regions and insulin biological activities, we designed a series of insulin analogues with changes at A21, A12-18 of C terminal helical region and A8-10 located in the region of A6-A11 intra-chain disulphide bond.METHODS:Insulin A-chain analogues were prepared by stepwise Fmoc solid phase manual synthesis and then combined with natural B-chain of porcine insulin to yield corresponding insulin analogues. Their biological activities were tested by receptor binding, mouse convulsion and immunological assay.RESULTS: A21Ala Ins retains 70.3% receptor binding capacity and 60% in vivo biological activity.DesA13-14, A21Ala Ins and DesA12-13-14-15, A21Ala Ins still have definite biological activity,7.9% and 4.0% receptor binding,and 6.2% and 3.3% in vivo biological activity respectively. A15Asn, A17Pro, A21Ala Ins maintains 10.4% receptor binding and 10% in vivo biological activity. A8His, A9Arg, A10Pro, A21Ala Ins, A8His, A9Lys, A10Pro, A21Ala Ins and A8His, A9Lys, A10Arg, A21Ala Ins have 51.9%, 44.3% and 32.1% receptor binding respectively,50%, 40% and 30% in vivo biological activity respectively, and 28.8%, 29.6% and 15.4% immunological activity respectively.CONCLUSION:A21Asn can be replaced by simple amino acid residues.The A chains with gradually damaged structural integrity in A12-18 helical region and the demolition of the A12-18 helical region by the substitution of Pro and Asn for A17Glu and A15Gln respectively can combine with the B chain and the combination products show definite biological activity, the helical structure of A12-18 is essential for biological activities of insulin. A8-10 is not much concerned with biological activities, but is much more important antigenically in binding to its antibodies, these results may help us design a new type of insulin analogue molecule.

Journal Article↗
PubMed2000

Cloning and Expression of Insulin Receptor Ligand-binding Domains.

Insulin receptor is a transmembrane protein consisting of four subunits, that form a heterotetramer(alpha(2)beta(2))with molecular weight of 350 kD. Because the extracellular subunit(alpha)consists of 731 residues and a cysteine-rich domain, it is difficult to express and crystallize such a large ligand-binding subunit, thus hampering further study on "insulin-receptor" complex. Based on the fact that the domains L1 and L2 of the alpha subunit, consisted of 119 and 118 residues, contained the high and low affinity insulin binding sites, respectively, the cDNAs of L1 and L2 were obtained from a human placental cDNA library by PCR. The cDNAs of L1, L2 and L1-(Ala)(10)-L2(designed ten-alanine-connected L1 and L2)were cloned, respectively, into an expression plasmid pET-3a, and E.coli BL21(DE3)transformants with such plasmids were successfully induced to express the goal proteins. The expression products were isolated and purified by the washing and solubilization of inclusion body, gel filtration chromatography and ion exchange chromatography. Each final product displayed a single band, corresponding the purity above 99%, in SDS-PAGE. These products have also been confirmed respectively as the L1, L2 and L1-(Ala)(10)-L2 by DNA sequencing, amino acid composition analysis and N-terminal amino acid sequencing.

Journal Article↗
PubMed2000

A Recombinant Monomeric Human Insulin Mutant with Resistance to Trypsin Design, Preparation and Characterization.

By using a gapped duplex DNA method, the four amino acids on the B chain of insulin, i.e. B22Arg, B28Pro, B29Lys and B30Thr, were mutagenized simultaneously into B22Asp, B28Lys, B29Pro and B30Lys, respectively. The recombinant B22Asp B28Lys B29Pro B30Lys human insulin was obtained simply by the treatment with trypsin of the precursor that was expressed in yeast. This human insulin analog had only 6% of receptor binding activity as that of the porcine insulin, but retained 50% of in vivo biological activity, compared with the latter. The self-association ability of the mutant measured through FPLC gel filtration chromatography showed that it was in monomeric state. As a monomeric insulin analog with resistance to the trypsin digestion, this compound may be promising in practical application.

Journal Article↗
PubMed2000

Possible Role of B8Gly in Insulin Structural Motif.

In the insulin structural motif n1-Cys-Gly-X10-Cys-n2-Cys-Cys-X3-Cys-X8-Cys-n3, there are seven absolutely conserved amino acid residues, and the only Gly is at position B8. When B8Gly was substituted with Ala by means of site-directed mutagenesis, a mutant insulin, [B8Ala]human insulin was obtained. The receptor binding capacity and in vivo biological activity of the [B8Ala]human insulin were about 2.5% and 10% of native porcine insulin, respectively. The far-UV circular dichroism (CD) spectra of [B8Ala]human insulin and human insulin showed that the relative content of alpha-helix in the mutant somewhat decreased. The results indicate that the B8Gly in the insulin structural motif is unreplaceble.

Journal Article↗
PubMed1999

Secretory Expression of Human Insulin in Methylotrophic Yeast Pichia pastoris.

The porcine insulin precursor (PIP) gene and its derivative form sp-PIP gene, which had a nona-peptide (called spacer peptide, sp) added at the 5' terminus of PIP gene, were inserted into the plasmid pPIC9 of Pichia pastoris to obtain secretory plasmid pPIC9/PIP and pPIC9/sp-PIP, respectively. P.pastoris GS115 was transformed by pPIC9/PIP or pPIC9/sp-PIP and the high-copy strains, P39(-sp) and S51(+sp), were selected by dot-blotting. The expression levels of PIP and sp-PIP were 10 mg/L and 40 mg/L in 1 L shake flask, respectively, indicating that the spacer peptide could increase the expression. The expression level of PIP (sp-PIP) in P.pastoris was higher than that of PIP in S.cerevisiae and K.lactis reported in this laboratory. The expression level of sp-PIP was 250 mg/L in 10 L fermentor. Recombinant human insulin was obtained by means of transpeptidation of PIP or sp-PIP. The receptor binding capacity is identical with that of porcine insulin. In vivo biological activity of the recombinant human insulin is 27 IU/mg.

Journal Article↗
PubMed1999

The Role of C-Terminus of Insulin B Chain and the Amino Group of B29Lys Side Chain in the Growth Promoting Activities of Insulin.

By growing the mouse mammary tumor-derived cell line GR2H6 in 96-well plates, we have developed an in vitro bioassay for the growth promoting activities of insulin. This bioassay system offers several advantages over currently used alternatives, such as higher sensitivity, better reproducibility and the processing of many samples simultaneously. Using this method, the mitogenic activities of insulin and its analogues were studied. The analogue with elongated C-tenminus of insulin B chain ( B31Lys Ins-NH(2)) had a higher mitogenic activity than insulin (130% of insulin). The mitogenic activities of analogues with B29Lys amino group blocked was one third of those with B29Lys amino group free, indicating that the C-terminal part of B chain and the amino group of B29Lys were important for the growth promoting activities of insulin.

Journal Article↗
PubMed1998

Comparison of the Growth Promoting Effects of Serum Transferrins from Different Animals on Mouse Mammary Tumor Cell Line GR2H6.

The growth promoting effects of seven animal serum transferrins from mammalian, aves, reptilia, amphibian and osteichthyes on mouse mammary tumor cell line GR2H6 in serum-free medium were compared by MTT assays. The results indicated that the mitogenic activities of the transferrins from different species were different, and this discrepancy was approximately parallel to their binding capacities with transferrin receptors on human placenta and GR2H6 cells.

Journal Article↗