کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1983201 1062351 2006 13 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Model reactions for insect cuticle sclerotization: Cross-linking of recombinant cuticular proteins upon their laccase-catalyzed oxidative conjugation with catechols
موضوعات مرتبط
علوم زیستی و بیوفناوری علوم کشاورزی و بیولوژیک دانش حشره شناسی
پیش نمایش صفحه اول مقاله
Model reactions for insect cuticle sclerotization: Cross-linking of recombinant cuticular proteins upon their laccase-catalyzed oxidative conjugation with catechols
چکیده انگلیسی

The quinone-tanning hypothesis for insect cuticle sclerotization proposes that N-acylcatecholamines are oxidized by a phenoloxidase to quinones and quinone methides, which serve as electrophilic cross-linking agents to form covalent cross-links between cuticular proteins. We investigated model reactions for protein cross-linking that occurs during insect cuticle sclerotization using recombinant pupal cuticular proteins from the tobacco hornworm, Manduca sexta, fungal or recombinant hornworm laccase-type phenoloxidase, and the cross-linking agent precursor N-acylcatecholamines, N-β-alanydopamine (NBAD) or N-acetyldopamine (NADA). Recombinant M. sexta pupal cuticular proteins MsCP36, MsCP20, and MsCP27 were expressed and purified to near homogeneity. Polyclonal antisera to these recombinant proteins recognized the native proteins in crude pharate brown-colored pupal cuticle homogenates. Furthermore, antisera to MsCP36, which contains a type-1 Rebers and Riddiford (RR-1) consensus sequence, also recognized an immunoreactive protein in homogenates of larval head capsule exuviae, indicating the presence of an RR-1 cuticular protein in a very hard, sclerotized and nonpigmented cuticle. All three of the proteins formed small and large oligomers stable to boiling SDS treatment under reducing conditions after reaction with laccase and the N-acylcatecholamines. The optimal reaction conditions for MsCP36 polymerization were 0.3 mM MsCP36, 7.4 mM NBAD and 1.0 U/μl fungal laccase. Approximately 5–10% of the monomer reacted to yield insoluble oligomers and polymers during the reaction, and the monomer also became increasingly insoluble in SDS solution after reaction with the oxidized NBAD. When NADA was used instead of NBAD, less oligomer formation occurred, and most of the protein remained soluble. Radiolabeled NADA became covalently bound to the MsCP36 monomer and oligomers during cross-linking. Recombinant Manduca laccase (MsLac2) also catalyzed the polymerization of MsCP36. These results support the hypothesis that during sclerotization, insect cuticular proteins are oxidatively conjugated with catechols, a posttranslational process termed catecholation, and then become cross-linked, forming oligomers and subsequently polymers.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Insect Biochemistry and Molecular Biology - Volume 36, Issue 4, April 2006, Pages 353–365
نویسندگان
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