Advanced

Characterization of an IgG glycan hydrolyzing enzyme from Streptococcus pneumoniae

Shadnezhad, Azadeh (2012) MOBN18 20112
Degree Projects in Molecular Biology
Abstract
Abstract

Streptococcus pneumonia secretes a number of glycosidases that modify the glycosylation state of immune defense molecules such as immunoglobulins. Modulation of the glycan on IgG can affect the function and structural properties on IgG and cause immune disorders. StrH is a β – N acetylglucosaminidase that removes terminally linked N-­‐ acetylglucosamines from human glycoproteins. It is described as an exoglycosidase, which cleaves sugar linkages after the activity of the exoglycosidases NanA and BgaA. In this study StrH was successfully isolated and activity of this enzyme on IgG, was investigated.

Popular science summary:

Streptococcus pneumonia (the pneumococcus) is a commensal of the human nasopharynx and extremely... (More)
Abstract

Streptococcus pneumonia secretes a number of glycosidases that modify the glycosylation state of immune defense molecules such as immunoglobulins. Modulation of the glycan on IgG can affect the function and structural properties on IgG and cause immune disorders. StrH is a β – N acetylglucosaminidase that removes terminally linked N-­‐ acetylglucosamines from human glycoproteins. It is described as an exoglycosidase, which cleaves sugar linkages after the activity of the exoglycosidases NanA and BgaA. In this study StrH was successfully isolated and activity of this enzyme on IgG, was investigated.

Popular science summary:

Streptococcus pneumonia (the pneumococcus) is a commensal of the human nasopharynx and extremely adapted to the mucosal surface of the respiratory tract. This bacterium can be an aggressive pathogen and cause diseases such as Otitis media, pneumonia, bacteremia and meningitis. More than 1 million deaths in the world is the result of pneumococcal infections each year.
Several factors such as glycosidases enable pneumococcus to evade recognition by the immune system or alter the responses directed against the infection. Human immune cells are glycosylated by different sugars and glycosidases are a group of enzymes that can cleave sugars linkages. Glycans are important for the function of glycoproteins and the removal or modification of the glycan by glycosidases can lead to a reduced or modified function of the protein. Pneumococcus has many glycosidases such as NanA, BgaA and StrH. These three enzymes can remove sugars from immune cells sequentially. In this project StrH was isolated and the glycosidase activity on IgG (Immunoglobulin G) was investigated to see the effect of this enzyme on the sugar chain of IgG. Investigation of hydrolase activity of StrH on IgG.
To isolate StrH, first, the DNA of pneumococcus was purified and the gene encoding StrH amplified by PCR (Polymerase Chain Reaction). A suitable vector was used (a vector is a circular small DNA for carrying the gene) and the vector with the gene of interest (strH) was transferred to another bacterium, Escherichia coli to express the enzyme.
The purified enzyme was incubated with IgG overnight at 37°C. Separation of the proteins by SDS PAGE and analysis of the glycans using glycan binding proteins, so called lectins, illustrated that SrtH is able to cleave sugar residues from the glycan of IgG.

Advisor: Mattias Collin
Master´s Degree Project 45 credits in Microbiology 2012
Department of infection medicine. Lund University (Less)
Please use this url to cite or link to this publication:
author
Shadnezhad, Azadeh
supervisor
organization
course
MOBN18 20112
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
3732479
date added to LUP
2013-04-30 13:40:01
date last changed
2013-04-30 13:40:01
@misc{3732479,
  abstract     = {Abstract

Streptococcus pneumonia secretes a number of glycosidases that modify the glycosylation state of immune defense molecules such as immunoglobulins. Modulation of the glycan on IgG can affect the function and structural properties on IgG and cause immune disorders. StrH is a β – N acetylglucosaminidase that removes terminally linked N-­‐ acetylglucosamines from human glycoproteins. It is described as an exoglycosidase, which cleaves sugar linkages after the activity of the exoglycosidases NanA and BgaA. In this study StrH was successfully isolated and activity of this enzyme on IgG, was investigated.

Popular science summary:

Streptococcus pneumonia (the pneumococcus) is a commensal of the human nasopharynx and extremely adapted to the mucosal surface of the respiratory tract. This bacterium can be an aggressive pathogen and cause diseases such as Otitis media, pneumonia, bacteremia and meningitis. More than 1 million deaths in the world is the result of pneumococcal infections each year.
Several factors such as glycosidases enable pneumococcus to evade recognition by the immune system or alter the responses directed against the infection. Human immune cells are glycosylated by different sugars and glycosidases are a group of enzymes that can cleave sugars linkages. Glycans are important for the function of glycoproteins and the removal or modification of the glycan by glycosidases can lead to a reduced or modified function of the protein. Pneumococcus has many glycosidases such as NanA, BgaA and StrH. These three enzymes can remove sugars from immune cells sequentially. In this project StrH was isolated and the glycosidase activity on IgG (Immunoglobulin G) was investigated to see the effect of this enzyme on the sugar chain of IgG. Investigation of hydrolase activity of StrH on IgG.
To isolate StrH, first, the DNA of pneumococcus was purified and the gene encoding StrH amplified by PCR (Polymerase Chain Reaction). A suitable vector was used (a vector is a circular small DNA for carrying the gene) and the vector with the gene of interest (strH) was transferred to another bacterium, Escherichia coli to express the enzyme. 
The purified enzyme was incubated with IgG overnight at 37°C. Separation of the proteins by SDS PAGE and analysis of the glycans using glycan binding proteins, so called lectins, illustrated that SrtH is able to cleave sugar residues from the glycan of IgG. 

Advisor: Mattias Collin 
Master´s Degree Project 45 credits in Microbiology 2012 
Department of infection medicine. Lund University},
  author       = {Shadnezhad, Azadeh},
  language     = {eng},
  note         = {Student Paper},
  title        = {Characterization of an IgG glycan hydrolyzing enzyme from Streptococcus pneumoniae},
  year         = {2012},
}