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Biology

Rock your Antibodies: Understanding an Ancient Immune System Protein in Brownbanded Bamboo Sharks

Sharks evolved into the top predators we know and love today millions of years ago. Because of their success at the top of the food chain, sharks have remained, in an evolutionary sense, relatively constant, meaning they haven’t evolved or changed much. This consistency provides shark scientists a window into the past, as some characteristics of modern sharks date back millions of years. One of the most prevalent examples of ancient characteristics still present in sharks today lies within the immune system.

    The immune system’s job is to prevent us from getting sick.  The system is made up of organs and proteins that prevent diseases from entering our bodies, kill diseases when they do enter, and prepare the body for their inevitable return. While there are many parts that make up this complex system, we will focus one key player: antibodies.

Antibodies are proteins with a very important job: to find pathogens, or intruders like bacteria and viruses and prevent them from entering your cells. Antibodies will bind to the pathogen, blocking its binding site so it can’t bind to your cells. Imagine a binding site like a lock and key – the pathogen is the key, and your cell has a lock. Antibodies cover the key so it no longer fits in the lock, and can’t open the door to your cell.  Instead, the pathogen, now covered in antibodies, will be consumed by an immune cell. The immune system will continue to work, completing multiple further steps until the pathogen is destroyed.

So, how do sharks play into this? Scientists have discovered that sharks have a very unique, ancient antibody.  To understand this, we must understand the structure of an antibody. In humans, antibodies are “Y” shaped, and made up of two chains of proteins: a heavy chain and a light chain (see figure 1). Sharks are unique, however, because they have an antibody made up of only one heavy chain – the light chain is completely absent.  Scientists believe this single-chain antibody, called the immunoglobulin novel antigen receptor (IgNAR, for short) represents an earlier stage in the evolution of the immune system. Because sharks have remained evolutionarily constant for millions of years, this small ancient relic still exists and functions in their bodies.

IgNAR antibodies are made up of one variable region, called V,  and 5 constant regions, C1-C5. The variable region is connected to the constant regions via a flexible bond that functions similar to a door hinge. This flexibility allows the antigen to stretch and contort in order to bind to intruding pathogens effectively. IgNAR is the smallest antibody discovered in any species.  The antibody’s small size and flexibility mean it could potentially be used for immunotherapy in humans, a type of treatment used to help our bodies fight diseases or cancer. However, in order to use IgNAR antibodies for treatment, we must first understand how they are made. This is where genetics comes into play.

As you may know, a gene is a long strand of DNA made up of a unique sequence of four bases (A, T, C, and G). Similar to how 26 letters can make millions of words, four bases can make millions of unique sequences.  These unique sequences are translated into RNA, which is like a template that codes for protein,s such as the proteins that make up IgNAR antibodies. By understanding which genes are translated to make IgNAR, we may someday be able to create IgNAR in a lab and use it to treat humans.

A study by De Silva et al. published in 2018 aimed to identify the genes that code for the different proteins that make up IgNAR. Although other studies have identified IgNAR genes in sharks before, the approach taken by De Silva et al. was unique. Previous studies have classified clusters of IgNAR genes based on the variable region (V) of the antibody. The gene encoding the C1 constant region, however, has been found to be more unique and have more variability than the other regions that make up IgNAR. The goal of this study was to classify IgNAR genes based on the C1 gene sequence, and to compare the IgNAR genes found in the bamboo shark to those found in other shark species. The C1 technique used in this study is novel, meaning this is the first time it has been used.

Methods

First, sharks reared at the Fisheries Laboratory at the University of Tokyo were anesthetized, or temporarily put to sleep, by spraying them with a mixture of chemicals. Then, scientists collected 8 ml of blood from each shark. Peripheral blood leukocytes (PBLs) were isolated from the blood samples. Peripheral blood leukocytes are white blood cells that circulate in your body and fight diseases. Antibodies are produced by a type of leukocyte called a B-cell, so the genes that code for IgNAR will be present in the PBL isolate.

Next, RNA was purified and isolated from the PBL samples. The RNA was then sequenced in order to construct a library consisting of all the RNA sequences present in the PBL sample. Once this library was prepared, scientists were ready to look at the sequences and determine which ones code for the proteins that  make up IgNAR. The IgNAR sequences from a nurse shark, which were found in a previous study, were used as a reference for the bamboo shark. The bamboo shark sequences were then assembled into a consensus sequence, and compared to sequences of other sharks.

Results

The bamboo shark library consisted of 20 million reads. That 20 million RNA sequences were read! Of these sequences, 154 were identified as IgNAR constant sequences by using the nurse shark sequences as a reference. These sequences were then combined to form two IgNAR sequences made up of 1758 base pairs.

The researchers found that brownbanded bamboo sharks have two types of IgNAR antibodies based on the C1 region: IgNAR-1 and IgNAR-2. In addition, the C1 region has 7 polymorphic sites. A polymorphic site is a position in a sequence that changes between individuals, for example, one bamboo shark might have a “C” base at position 112, while another bamboo shark has an “A” in that position (think poly=many, morph=change). Based on the different bases found in the polymorphic sites, the researchers determined that there were 13 unique possibilities for C1 gene sequences present in bamboo sharks.

When compared to other sharks’ IgNAR sequences, the researchers found that brownbanded bamboo sharks were 77% similar to nurse sharks and more than 57% similar to other cartilaginous fish. The IgNAR genes were also found to have some similarities to human antibody genes.

 

Why do we care?

Sharks have very unique immune systems. Despite inhospitable environments for immune cells, such as high concentrations of the toxic element urea in shark blood, sharks rarely get diseases and don’t get cancer. This means that shark immune cells must be especially suited to handle harsh environments and fight off diseases. Someday, we may be able to use our knowledge of shark immunity to treat human diseases and ailments. Before we reach that point, however, we must first understand the basics of shark immunology by identifying the genes that make up immune system proteins.

In addition, this study was the first to present a unique method of categorization of IgNAR antibodies using the C1 constant region. This region’s variability make it more suitable for classification than other regions of IgNAR. This finding will allow scientists to replicate the methods in this study to make further discoveries. The research presented here brings us one step closer to solving the mysteries of immunology, one shark at a time.

 

Source:

D.P.N. De Silva, E. Tan, N. Mizuno, S. Hosoya, Md S. Reza, S. Watabe, S. Kinoshita, S. Asakawa, Transcriptomic analysis of immunoglobulin novel antigen receptor (IgNAR) heavy chain constant domains of brownbanded bamboo shark (Chiloscyllium punctatum), Fish & Shellfish Immunology, Volume 84, 2019, Pages 370-376, ISSN 1050-4648, https://doi.org/10.1016/j.fsi.2018.10.004. (http://www.sciencedirect.com/science/article/pii/S1050464818306284)

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