Molecular basis of the inhibition of βs-chain-dependent polymerization by mouse α-chain: Semisynthesis of chimeras of human and mouse α-chains

Rajendra P. Roy, Ronald L. Nagel, A. Seetharama Acharya

Research output: Contribution to journalArticle

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Abstract

The transgenic mouse models expressing βs-globin genes do not fully exhibit the sickling phenotype, primarily as a result of the inhibition of βs-chain-dependent polymerization by the mouse α-chains. The mouse α-chain differs from the human α-chain at 19 sequence locations. Of these, only α78 and α116 are the known hemoglobin (Hb) S polymer contact sites. To define whether the inhibition of polymerization by the mouse α-chain is solely a consequence of the differences at these two sites or additional sites of sequence differences are also involved, we have constructed chimeric α-chains by employing the α-globin semisynthetic reaction (Sahni, G., Cho, Y. J., Iyer, K. S., Khan, S. A., Seetharam, R., and Acharya, A. S. (1989) Biochemistry 28, 5456-5461). Mouse α1-30 was spliced with human α31-141 using endoproteinase Glu-C to generate a chimeric α-globin (αMH) containing eight of the 19 sequence differences of mouse α-globin. Similarly, human α1-30 was spliced with mouse α31-141 to generate another chimeric α-globin (αHM) containing 11 sequence differences. The respective chimeric globins were purified, reconstituted with heme and βs-chain into tetrameric hemoglobin, and the tetramers were purified by ion-exchange chromatography. The inhibitory potential of the chimeric αMH-chain on the polymerization is 10-fold lower than that of the mouse α-chain. The absence of the α31-141 region of the mouse α-chain relieves only a portion of the inhibition. The inhibitory potential of αMH contributed by the mouse α1-30 segment is significant although none of the sequence differences in this segment are located at any of the implicated polymer contact sites. The chimeric αHM-chain also inhibits the polymerization, but the extent of inhibition is again lower (4-fold) than that of the full-length mouse α-chain. The results demonstrate that the inhibitory potential of mouse α-chains involves the sequence differences from both the α1-30 and α31-141 regions. Besides, since the sum of the inhibitory potential of either of these chimeric α-chains is lower than that of the intact mouse α-chains, we speculate that conformational changes that require the copresence of sequence differences in both portions of the mouse α-chain also contribute to the inhibitory propensity of the mouse α-chain.

Original languageEnglish (US)
Pages (from-to)16406-16412
Number of pages7
JournalJournal of Biological Chemistry
Volume268
Issue number22
StatePublished - Aug 5 1993

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Globins
Polymerization
Polymers
Sickle Hemoglobin
Biochemistry
Chromatography
Heme
Ion exchange
Hemoglobins
Genes
Ion Exchange Chromatography
Transgenic Mice

ASJC Scopus subject areas

  • Biochemistry

Cite this

Molecular basis of the inhibition of βs-chain-dependent polymerization by mouse α-chain : Semisynthesis of chimeras of human and mouse α-chains. / Roy, Rajendra P.; Nagel, Ronald L.; Acharya, A. Seetharama.

In: Journal of Biological Chemistry, Vol. 268, No. 22, 05.08.1993, p. 16406-16412.

Research output: Contribution to journalArticle

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abstract = "The transgenic mouse models expressing βs-globin genes do not fully exhibit the sickling phenotype, primarily as a result of the inhibition of βs-chain-dependent polymerization by the mouse α-chains. The mouse α-chain differs from the human α-chain at 19 sequence locations. Of these, only α78 and α116 are the known hemoglobin (Hb) S polymer contact sites. To define whether the inhibition of polymerization by the mouse α-chain is solely a consequence of the differences at these two sites or additional sites of sequence differences are also involved, we have constructed chimeric α-chains by employing the α-globin semisynthetic reaction (Sahni, G., Cho, Y. J., Iyer, K. S., Khan, S. A., Seetharam, R., and Acharya, A. S. (1989) Biochemistry 28, 5456-5461). Mouse α1-30 was spliced with human α31-141 using endoproteinase Glu-C to generate a chimeric α-globin (αMH) containing eight of the 19 sequence differences of mouse α-globin. Similarly, human α1-30 was spliced with mouse α31-141 to generate another chimeric α-globin (αHM) containing 11 sequence differences. The respective chimeric globins were purified, reconstituted with heme and βs-chain into tetrameric hemoglobin, and the tetramers were purified by ion-exchange chromatography. The inhibitory potential of the chimeric αMH-chain on the polymerization is 10-fold lower than that of the mouse α-chain. The absence of the α31-141 region of the mouse α-chain relieves only a portion of the inhibition. The inhibitory potential of αMH contributed by the mouse α1-30 segment is significant although none of the sequence differences in this segment are located at any of the implicated polymer contact sites. The chimeric αHM-chain also inhibits the polymerization, but the extent of inhibition is again lower (4-fold) than that of the full-length mouse α-chain. The results demonstrate that the inhibitory potential of mouse α-chains involves the sequence differences from both the α1-30 and α31-141 regions. Besides, since the sum of the inhibitory potential of either of these chimeric α-chains is lower than that of the intact mouse α-chains, we speculate that conformational changes that require the copresence of sequence differences in both portions of the mouse α-chain also contribute to the inhibitory propensity of the mouse α-chain.",
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N2 - The transgenic mouse models expressing βs-globin genes do not fully exhibit the sickling phenotype, primarily as a result of the inhibition of βs-chain-dependent polymerization by the mouse α-chains. The mouse α-chain differs from the human α-chain at 19 sequence locations. Of these, only α78 and α116 are the known hemoglobin (Hb) S polymer contact sites. To define whether the inhibition of polymerization by the mouse α-chain is solely a consequence of the differences at these two sites or additional sites of sequence differences are also involved, we have constructed chimeric α-chains by employing the α-globin semisynthetic reaction (Sahni, G., Cho, Y. J., Iyer, K. S., Khan, S. A., Seetharam, R., and Acharya, A. S. (1989) Biochemistry 28, 5456-5461). Mouse α1-30 was spliced with human α31-141 using endoproteinase Glu-C to generate a chimeric α-globin (αMH) containing eight of the 19 sequence differences of mouse α-globin. Similarly, human α1-30 was spliced with mouse α31-141 to generate another chimeric α-globin (αHM) containing 11 sequence differences. The respective chimeric globins were purified, reconstituted with heme and βs-chain into tetrameric hemoglobin, and the tetramers were purified by ion-exchange chromatography. The inhibitory potential of the chimeric αMH-chain on the polymerization is 10-fold lower than that of the mouse α-chain. The absence of the α31-141 region of the mouse α-chain relieves only a portion of the inhibition. The inhibitory potential of αMH contributed by the mouse α1-30 segment is significant although none of the sequence differences in this segment are located at any of the implicated polymer contact sites. The chimeric αHM-chain also inhibits the polymerization, but the extent of inhibition is again lower (4-fold) than that of the full-length mouse α-chain. The results demonstrate that the inhibitory potential of mouse α-chains involves the sequence differences from both the α1-30 and α31-141 regions. Besides, since the sum of the inhibitory potential of either of these chimeric α-chains is lower than that of the intact mouse α-chains, we speculate that conformational changes that require the copresence of sequence differences in both portions of the mouse α-chain also contribute to the inhibitory propensity of the mouse α-chain.

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