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Investigation of Sushi Domain-Containing Protein 4 (SUSD4) and its effect on INS-1 832/13 cells

Danestig, Karolina (2017) MOBT01 20162
Degree Projects in Molecular Biology
Abstract
Type 2 diabetes is becoming an increasingly more common disease in the world, with a strong correlation to increasing obesity. Previous studies have shown that obese patients have an increased level of inflammatory mediators believed to be one of the underlying causes for diabetes development. One part of the immune system, contributing to these mediators, is the complement system. Investigating the role complement system plays in the development of diabetes is therefore of interest. Recently, a new protein was discovered restraining the complement system called Sushi Domain-Containing Protein 4 (SUSD4). Not much is known about this complement inhibitor, but the fact that it is expressed in human pancreatic beta cells rises the question if... (More)
Type 2 diabetes is becoming an increasingly more common disease in the world, with a strong correlation to increasing obesity. Previous studies have shown that obese patients have an increased level of inflammatory mediators believed to be one of the underlying causes for diabetes development. One part of the immune system, contributing to these mediators, is the complement system. Investigating the role complement system plays in the development of diabetes is therefore of interest. Recently, a new protein was discovered restraining the complement system called Sushi Domain-Containing Protein 4 (SUSD4). Not much is known about this complement inhibitor, but the fact that it is expressed in human pancreatic beta cells rises the question if SUSD4 could influence insulin secretion and therefore, also the development of diabetes. In this study, SUSD4 knock out INS-1 832/13 cells were created using the CRISPR-Cas9 system. The knock out clones’ ability to secrete insulin was tested and compared to wild type, with a result suggesting that SUSD4 has no significant effect on insulin secretion in vitro. This report also describes two attempts of generating an antibody against mouse SUSD4, hoping to study the protein further in vivo. Here, generation and purification of both a rabbit antibody against recombinant mouse SUSD4 and a rabbit antibody against a SUSD4 peptide are described. This report also contains testing of the antibodies and propositions on how an antibody can be generated against mouse SUSD4 in the future. (Less)
Popular Abstract
Study of a newly discovered protein and its possible role in diabetes

When you get sick due to a pathogen (virus or bacteria) invasion, it is important that your body can defeat the infection. This is the reason we have an immune system taking care of us. Even though the immune system works to keep you healthy, it might destroy your own cells and tissues when fighting the infection. The immune system is therefore strictly controlled to keep the immune response at a level that will kill the pathogen without harming you. One protein helping with controlling the immune response, by restraining it, is the Sushi Domain-Containing Protein 4 (SUSD4).

SUSD4 is a recently discovered protein, which is the reason why not much is known about... (More)
Study of a newly discovered protein and its possible role in diabetes

When you get sick due to a pathogen (virus or bacteria) invasion, it is important that your body can defeat the infection. This is the reason we have an immune system taking care of us. Even though the immune system works to keep you healthy, it might destroy your own cells and tissues when fighting the infection. The immune system is therefore strictly controlled to keep the immune response at a level that will kill the pathogen without harming you. One protein helping with controlling the immune response, by restraining it, is the Sushi Domain-Containing Protein 4 (SUSD4).

SUSD4 is a recently discovered protein, which is the reason why not much is known about the protein and its functions in our body. One thing we know is that the protein can be found in pancreatic beta cells. Beta cells are of importance in the pancreas since they secrete insulin. Insulin gives signals to cells in the body to take up glucose (sugar) from the blood and create energy essential for your body to work. We also know that patients with pancreatic diseases such as diabetes (an insulin secretion deficiency) often show signs of inflammation, which SUSD4 might prohibit. By studying the role of SUSD4 in insulin secretion we aim to find out more about the SUSD4 function and its possible role in diabetes development.

SUSD4 had no effect on insulin secretion in cultured rat beta cells
In this project, we removed (knock out) SUSD4 from cultured rat beta cells by genetically modifying them and creating SUSD4 knock out cells. We then investigated the insulin secretion ability of these SUSD4 knock out cells by exposing them to a high glucose concentration after being in a low glucose concentration. This is basically what happens to the beta cells in your body after a meal. We compared the amount of insulin secreted from the knock out cells to the level secreted by normal cells (wild type cells).

The amount of insulin secreted was analysed using an Enzyme-linked immunosorbent assay (ELISA). ELISA is a method used to detect proteins, using labelled antibodies that bind to the protein of interest (here: insulin). The label on the antibody can be detected using a spectrophotometer; the amount of antibody detected in the sample is proportional to the insulin level in the sample. This way, the amount insulin secreted from the cells can be measured.

The graph shows that there is no significant difference in insulin secretion between wild type and SUSD4 knock out cells. Though, one must consider that this experiment was only performed on cells without the complex influence of signals from other cells in the body. The result might therefore differ when looking at insulin secretion in the whole body. This result is a step forward towards better understanding of the function of SUSD4.

Master’s Degree Project in Molecular Biology, 60 credits 2017
Department of Biology, Lund University

Advisor: dr. Wouter Van Overbeke and prof. Anna Blom
Medical Protein Chemistry, Department of Translational Medicine, Lund University, Malmö (Less)
Please use this url to cite or link to this publication:
author
Danestig, Karolina
supervisor
organization
course
MOBT01 20162
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
8905379
date added to LUP
2017-04-03 16:08:32
date last changed
2017-04-03 16:08:32
@misc{8905379,
  abstract     = {Type 2 diabetes is becoming an increasingly more common disease in the world, with a strong correlation to increasing obesity. Previous studies have shown that obese patients have an increased level of inflammatory mediators believed to be one of the underlying causes for diabetes development. One part of the immune system, contributing to these mediators, is the complement system. Investigating the role complement system plays in the development of diabetes is therefore of interest. Recently, a new protein was discovered restraining the complement system called Sushi Domain-Containing Protein 4 (SUSD4). Not much is known about this complement inhibitor, but the fact that it is expressed in human pancreatic beta cells rises the question if SUSD4 could influence insulin secretion and therefore, also the development of diabetes. In this study, SUSD4 knock out INS-1 832/13 cells were created using the CRISPR-Cas9 system. The knock out clones’ ability to secrete insulin was tested and compared to wild type, with a result suggesting that SUSD4 has no significant effect on insulin secretion in vitro. This report also describes two attempts of generating an antibody against mouse SUSD4, hoping to study the protein further in vivo. Here, generation and purification of both a rabbit antibody against recombinant mouse SUSD4 and a rabbit antibody against a SUSD4 peptide are described. This report also contains testing of the antibodies and propositions on how an antibody can be generated against mouse SUSD4 in the future.},
  author       = {Danestig, Karolina},
  language     = {eng},
  note         = {Student Paper},
  title        = {Investigation of Sushi Domain-Containing Protein 4 (SUSD4) and its effect on INS-1 832/13 cells},
  year         = {2017},
}