Галектин-опосредованное связывание вируса гриппа с клетками-мишенями тема диссертации и автореферата по ВАК РФ 02.00.10, кандидат химических наук Черный, Евгений Станиславович

  • Черный, Евгений Станиславович
  • кандидат химических науккандидат химических наук
  • 2013, МоскваМосква
  • Специальность ВАК РФ02.00.10
  • Количество страниц 170
Черный, Евгений Станиславович. Галектин-опосредованное связывание вируса гриппа с клетками-мишенями: дис. кандидат химических наук: 02.00.10 - Биоорганическая химия. Москва. 2013. 170 с.

Оглавление диссертации кандидат химических наук Черный, Евгений Станиславович

СОДЕРЖАНИЕ

Стр

Список использованных сокращений

1 ВВЕДЕНИЕ

2 ОБЗОР ЛИТЕРАТУРЫ

Глава 1 Структурные компоненты вируса гриппа

1 1 Структура нейраминидазы 1 2 Гликозилирование нейраминидазы 1 3 Структура гемагглютинина

1 4 Гликозилирование гемагглютинина

Глава 2 Сборка и отпочковывание (баддинг) вируса гриппа

2 1 Липидный состав вируса гриппа

2 2 Роль НА и ИА в сборке и баддинге вирионов

2 3 Роль М1 и М2 в сборке и баддинге вирионов

Глава 3 Структурная организация вируса гриппа

3 1 Современные методы изучения морфологии вируса гриппа 3 2 Размеры и форма вирионов

3 3 Поверхность вируса гриппа

Глава 4 Галектины

4 1 Структура галектинов

4 2 Биологическая роль галектинов 4 3 Куриные галектины

3 МАТЕРИАЛЫ И МЕТОДЫ

4 РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ

1 Проверка первых двух гипотез Роль белков в составе вириона

1 1 Поиски белков клетки-хозяина

1 2 Поиск «неклассических» углеводных рецепторов

для вируса гриппа

2 Взаимодействие вируса гриппа с галектинами

2 1 Изучение связывания вируса гриппа с галектинами

с помощью твердофазного анализа 2 2 Влияние галектинов на функционирование НА и

КА вируса гриппа 3 Ингибирование галектинами связывания антител с вирусом

3 1 Ингибирование связывания поликлональных

антител с вирусом гриппа 3 2 Ингибирование галектинами связывания сыворотки больных гриппом с вирусом гриппа

3 3 Ингибирование галектинами связывания

внутривенного иммуноглобулина с вирусом гриппа 4 Роль галектинов на стадии адгезии вирионов на клетке

4 1 Расположение галектинов и вируса гриппа на поверхности клетки 4 2 Изучение влияния галектинов на связывание вируса гриппа

с клетками

4 3 Проверка влияния флуоресцентной метки на связывание

вируса с галектинами 4 4 Действие галектинов при нагрузке на вирионы 4 5 Ингибирование взаимодействия галектинов с вирусом 4 6 Влияние на адгезию гликома вируса и клеточной поверхности

4 7 Связывание вируса с десиалилированными клетками

в присутствии галектинов

5 Мечение вирусов гриппа с помощью флуоресцентного липида

127

6 Исследование полного цикла заражения вируса в присутствии галектинов 6 1 Влияние галектинов на заражение монослоя клеток вирусом 6 2 Действие галектинов на эффект заражения клеток вирусом гриппа,

132

сравнение разных условий проведения эксперимента

7 О возможности участия галектинов в инфекционном процессе вируса гриппа

142

5 ЗАКЛЮЧЕНИЕ

6 ВЫВОДЫ

7 Благодарности

8 СПИСОК ЛИТЕРАТУРЫ

146

147

148

Список использованных сокращений

АЭК - аминоэтилкарбазол FITC - флуоресцеинизотиоционат ДСН - додецилсульфат натрия Fluo-DOPE - флуоресцентный липид ГАЕ - гемагглютинирующая единица М - матриксный белок НА - гемагглютинин

НВ-сайт - гемоадсорбционный сайт нейраминидазы РГА - реакция гемагглютинации ТФА - твердофазный анализ РНП - рибонуклеопротеин

MDCK - линия клеток Madin-Darby Canine Kidney

NA - нейраминидаза

NP - нуклеопротеин

NS - неструктурный белок

CG - Куриные галектины

hG - Человеческие галектины

УСД - углевод связывающий домен

Сахариды, конъюгаты и субстраты NA:

Glyc-PAA-biot - биотинилированный гликоконъюгат конъюгат на основе полиакриламида

Neu5Ac - N-ацетилнейраминовая кислота

3'SL - Neu5Aca2-3Gaipi-4Glc

3'SLN - Neu5Aca2-3Gaipi-4GlcNAc

6'SLN - Neu5Aca2-6Gaipi-4GlcNAc

Lex - Gaipi-4(Fucal-3)GlcNAcp

B,ri- Galal-3(Fucal-2)Galp

Fs-2 - GalNAcal-3GalNAcp

Tn - aGalNAc

LacNAc - Gaipi-4GlcNAc

А тип 1 -GalNAcal-3(Fucal-2)Galpl-3GlcNAc

А тип 2 - GalNAcal -3(Fuca 1 -2)Gaip 1 -4GlcNAc

В тип 1 - Gala 1 -3 (Fuca 1 -2)Gaip 1 -3 GlcNAc

В тип 2 - Galal-3(Fucal -2)Gaipi -4GlcNAc

H тип 1 -Fucal-2Gaipi-3GlcNAc

H тип 2 - Fucal-2Gaipi-4GlcNAc

MU-NANA - метилумбеллиферил N-ацетилнейраминовая кислота BODIPY - 6-((4,4-дифторо-5,7-диметил-4-бора-За,4а-даиза-5-индацин-3-пропионил)амино)гексаноилвая кислота сукцинимидный эфир

Рекомендованный список диссертаций по специальности «Биоорганическая химия», 02.00.10 шифр ВАК

Введение диссертации (часть автореферата) на тему «Галектин-опосредованное связывание вируса гриппа с клетками-мишенями»

1. ВВЕДЕНИЕ

Вирус гриппа - один из наиболее изученных объектов среди возбудителей опасных заболеваний человека. Тем не менее, в начале 21 века человечество фактически не имеет ни надежного лекарства против него, ни системы вакцинации, способной гарантированно предотвратить заболевание. Поэтому расширение представлений о структурной организации, рецепторной специфичности и репликации вируса в организме хозяина, с целью разработки принципиально новых подходов к терапии и предупреждению этого заболевания, является крайне актуальной задачей.

Стадии первичного связывания и проникновения вируса гриппа в заражаемую клетку по-прежнему являются предметами активного изучения. На сегодняшний день, устоявшиеся представления о том, что единственным рецептором вируса гриппа являются сиалогликаны, регулярно подвергаются сомнению, и высказываются предположения о существовании дополнительных факторов, участвующих в адгезии вирионов на поверхности клетки (Stray and others 2000; Rapoport and others 2006; M L Yang and others 2011). В связи с этим, исследования в данном направлении являются актуальной задачей, решение которой может привести к разработке нового поколения лекарственных препаратов - блокаторов адгезии вируса гриппа. Кроме того, детальное изучение комплексных процессов, протекающих в организме при иммунном ответе на вторжение вируса, и, прежде всего, реакция к поверхностным гликопротеинам вирионов как антигенам, открывает возможности для разработки новых иммунотерапевтических подходов.

Цели и задачи исследования.

Целью данной кандидатской диссертации являлось обнаружение дополнительных факторов, участвующих в рецепции вируса гриппа на начальной стадии заражения клеток.

Предполагалось решить следующие задачи:

1. Изучить возможность связывания вируса гриппа с несиалилированными углеводными лигандами.

2. Экспериментально проверить гипотезу о возможности связывания галектинов с вирусом гриппа и установить роль этих белков во взаимодействии вирусных частиц с клеткой-мишенью.

3. Изучить влияние галектинов на связывание антител с вирусом гриппа.

Научная новизна.

• большая часть представителей семейства куриных и человеческих галектинов специфично связываются с вирусом гриппа;

• галектины, несмотря на способность увеличивать адгезию вируса

5

гриппа на клетке-мишени, выступают как ингибиторы процесса заражения;

• при отсутствии основного сиало-рецептора, галектины могут обеспечивать высокий уровень адгезии вируса на клетке;

• связывание галектинов с вирусом гриппа не приводит к изменению функциональных свойств гемагглютинина и нейраминидазы, но маскирует их антигенные детерминанты.

Практическая значимость.

^ Обнаруженная компенсаторная роль галектинов при отсутствии сиалорецепторов на клетке заставляет по-новому относиться к терапевтическим методам, нацеленным на ингибирование сиало-опосредованного заякоревания вируса гриппа, а именно, учитывать этот фактор при испытании антиадгезионных препаратов.

^ Галектин-опосредованную адгезию вируса гриппа на клетке необходимо учитывать в производстве живых вакцин, которые получают наращиванием вируса в клеточных культурах.

^ Найденный эффект ингибирования галектинами протективных антивирусных антител открывает новую возможную область для совершенствования анти-вирусной иммунотерапии.

2. ОБЗОР ЛИТЕРАТУРЫ

Похожие диссертационные работы по специальности «Биоорганическая химия», 02.00.10 шифр ВАК

Заключение диссертации по теме «Биоорганическая химия», Черный, Евгений Станиславович

6. выводы

1. Обнаружена способность галектинов связываться с вирусом гриппа. Нагрузка галектинов на поверхность вируса не изменяет функциональную активность гемагглютинина и нейраминидазы.

2. Связывание с галектинами маскирует антигенные детерминанты поверхности вируса гриппа, предохраняя его от присоединения антител.

3. Галектины в отсутствие сиалозидов способны обеспечивать адгезию вируса гриппа на клетке-мишени. На галектин-опосредованную адгезию влияет углеводный состав как вируса, так и клетки.

4. Галектины ингибируют инфицирование, несмотря на то, что способствуют адгезии вируса гриппа на клетке.

5. Предложен новый метод мечения вируса гриппа с помощью введения гидрофобной флуоресцентной метки в состав липидной мембраны вирионов.

7. Благодарности

Автор крайне признателен A.C. Гамбарян (Институт полиомиелита и вирусных энцефалитов им. М.П.Чумакова РАМН, Москва) за помощь в выращивании вирусов, Е.М. Рапопорт (ИБХ РАН, Москва) за помощь в клеточной работе, Е.Ю. Корчагиной (ИБХ РАН, Москва) за синтез Fluo-DOPE и обсуждение работы, Л.В. Кордюковой (НИИ ФХБ, Москва) за рекомендации в проведении ряда экспериментов, Т. Гардеру (Институт Фридриха-Леффлера, Грайфсвальд, Германия) за сотрудничество в проведении ряда экспериментов, М.Н. Матросовичу (Университет имени Филиппа, Марбург, Германия) за предоставление культуры MDCK6SIAT, Е.И. Бурцевой (НИИ Вирусологии, Москва) за предоставление вируса H1N1 Москва и человеческой сыворотки и Г.-И. Габиусу (Университет имени Максимилиана, Мюнхен, Германия) за предоставление галектинов.

5. ЗАКЛЮЧЕНИЕ

Несмотря на то, что вирус гриппа является одним из самых хорошо изученных вирусов, он, по-прежнему, представляет опасность для здоровья человека. Лекарственные препараты, которые доступны на сегодняшний день, направлены на подавление активности нейраминидазы или М2 белка вируса гриппа. Между тем, эти компоненты являются минорными, в то время как большую часть поверхности вируса занимает гемагглютинин. Однако, лекарственных средств направленного действия против гемагглютинина пока не существует. Выявленные в данной работе закономерности делают более полным понимание процесса адгезии вируса на клетке-мишени. Полученные результаты могут способствовать разработке терапевтического подхода, объединяющего блокирование гемагглютинина и галектина. Еще одним средством борьбы с вирусом гриппа является вакцинация. Несмотря на огромные усилия по разработке синтетических вакцин, реально действующими и надежными остаются вакцины, основанные на целых вирионах, которые по современным технологиям нарабатываются в клеточной культуре. Обнаруженные в данном исследовании факты влияния галектинов на адгезию вирусов к клетке и на процесс заражения, несомненно, внесут определенные коррективы в будущие разработки новых противогриппозных вакцин.

Список литературы диссертационного исследования кандидат химических наук Черный, Евгений Станиславович, 2013 год

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