Scientists Identify the Target of an Immune Suppression Molecule Called CD22—Itself
A New Technique for Looking at Sugar-Binding Proteins

By Jason Socrates Bardi

Scientists at The Scripps Research Institute have applied an innovative approach to studying human proteins that bind to sugar molecules on the surface of human cells to discover how one crucial aspect of our immune system works. They identified the target of a regulatory molecule called CD22 that is expressed on the surface of the type of immune system cell known as the B lymphocyte.

B lymphocytes, the "B" of which comes from the fact that they are created in our "bone" marrow, are one of the most important immune cells in our bodies. One of the two major types of immune cells in the adaptive immune system, they play a critical role in the body's ability to fight off infection because they produce antibodies, which are a crucial component of the adaptive immune response to bacteria and viruses that successfully invade the body.

But B cells, like all the components of the immune system, must be kept in check because B cell activation initiates drastic physiological measures such as inflammation and cell killing. These actions are great for killing off unwanted bacteria and viruses, but they can also harm healthy human tissue. So over time humans and other mammals have evolved systems for ensuring that B cells are only activated and set loose when absolutely necessary.

For years, scientists have known that B cells have a self-regulatory mechanism that involves CD22 molecules on the cell surface, like a dampening switch that keeps B cells from becoming active. But scientists did not know how the molecule does so. Part of the problem was that they did not know the target of CD22.

Now that part of the mystery has been solved. According to a paper in an upcoming issue of the journal Nature Chemical Biology, the molecule CD22 targets itself.

This is a crucial first step in understanding the complete picture of how CD22 and B cell activation work, says Professor James Paulson, who led the research with his colleagues in the Department of Molecular Biology at Scripps Research. "Understanding these mechanisms will be important for coming up with new ways of addressing immunologic disorders [that involve B cell activation]."

Like a Wad of Stolen Cash

B lymphocytes are like those brightly colored explosive dye packs that bank tellers carefully tuck into stacks of bills handed over to unwary bank robbers. Once the dye pack is out of the bank's walls it gets activated. Then it explodes and covers the rest of the stolen bills in the sack with a telltale fluorescent dye.

Similarly, B cells, once they are alerted to the presence of some pathogen in the body, will become active, proliferate, and release gobs of antibody proteins that then alert the immune system to the presence of the invaders, attract lethal "effector" immune cells to the site of infection, block viruses or bacteria from infecting host cells, and mark those bacteria or viruses for destruction by other immune system cells, such as macrophages.

A lot is known about B cells. They arise in the bone marrow and develop into mature inactive cells that circulate through the bloodstream. These mature B cells express one particular type of B-cell receptor, and they have the ability to produce one particular type of antibody (which is really just a secreted form of this B-cell receptor). The B-cell receptor is displayed on the surface of the B cell, where it comes into contact with antigens—proteins, carbohydrate molecules, and other pieces of a foreign bacterium, virus, or other pathogen.

Many B cells in the body are designed to recognize a wide range of foreign antigens, and countless numbers of these may encounter any particular antigen that appears during an infection. As essential as it is to activate these B cells and protect us against infection in a world rotten with pathogens, it's also quite dangerous. Active B cells can cause a lot of collateral damage to healthy tissue, and one would not want to activate B cells unnecessarily any more than a bank teller would want to give away exploding ink packs to honest customers.

So the body has ways of keeping the B cells in the bottom of the drawer until a thief comes along. One basic way the body does this is by setting a threshold for B cell activation. Only those B cells with receptors that can bind to the antigen with a certain affinity will become activated, undergo clonal expansion, and begin producing antibody.

The body also has built-in safety mechanisms that continually shut off the signals that lead to B cell activation. One of the molecules involved in this regulatory process is the accessory protein CD22, which is expressed on the surface of B cells. CD22 acts as a safety valve of sorts, preventing the accidental or aberrant activation of B cells.

Evidence for CD22's role, says Paulson, can be seen in mice that have no CD22 molecules. These mice have a hyperactive immune system and are prone to developing spontaneous autoimmune disease.

What We Know and Don't Know About How CD22 Works

When a B-cell receptor encounters the antigen for which it is specific, the recognition of this antigen by the B cells' receptors initiates a complicated cascade of "signaling" events that leads to the B cells' activation. An early part of this signaling involves the action of enzymes known as kinases, which attach chemical groups known as phosphates onto the B-cell receptor and its associated proteins that are on the inside of the cell membrane.

The degree to which these phosphates are attached determines whether the signaling cascade will continue and whether B cells will become activated or not. Since the attachment of these phosphates regulates B cell activation, their detachment downregulates B cell activation. This is where CD22 comes in.

It, like the B-cell receptor, is an integral membrane protein and has surfaces on both sides of the B cell membrane. On the inside of the membrane is a domain that recruits an enzyme known as a phosphatase. The action of the phosphatase is to remove phosphate groups from other proteins—such as the B-cell receptor complex of proteins.

And CD22 will constantly recruit these phosphatases and downregulate B cell activation unless it is itself shut off. Exactly how CD22 is shut off (thus enabling the activation of the B cell) has been of interest to scientists for several years.

While the business end of the CD22 that recruits the phosphatase and keeps the B cell in check is on the inside of the cell membrane, the trigger that shuts CD22 off appears to be on the outside of the membrane. There, on the outside of the cell, the CD22 molecule has a domain that recognizes a particular type of sugar called sialic acid. Once CD22 binds to its target sialic acid, it is pulled away from the B-cell receptor. Then the B-cell receptor is free to be phosphorylated.

Along with Jamey Marth at the University of California, San Diego, Paulson and colleagues showed the importance of this sugar a few years ago when they demonstrated that mice lacking the enzyme that makes the sugar are severely immunosuppressed. Since their CD22 molecules are never "distracted" from recruiting the phosphatase that constantly shuts down the B-cell receptor signaling, the B cells are poorly activated.

Scientists knew that CD22 was binding to sialic acid in the context of another protein on the surface of the B cell. The question was, what was this target glycoprotein? This has not been so straightforward to answer, says Paulson. "The organization of the cell surface is more complex than you might think."

Cells in the body are covered with glycoproteins, and B cells are no exception. One of the most common sugars decorating glycoproteins is the sialic acid to which CD22 binds. It has been difficult to identify the particular target of CD22 because there are many possibilities—in fact the list of potential candidates is almost every glycoprotein on the B cell.

Hoping to narrow this list down, scientists have tried to determine experimentally which glycoproteins CD22 binds to, but until recently their success was limited. It's nearly impossible to separate CD22 from the rest of the contents of the cell without also stripping away whatever CD22 is bound to.

Careful experiments in which CD22 was attached to an antibody and allowed to bind to different cellular glycoproteins in vitro yielded inconclusive results—the experiments identified not one but a number of glycoproteins to which CD22 can bind. This stymied scientists because they did not know which of all these possible targets CD22 bound to during the regulation of B cell activation.

For instance, the B cell surface proteins CD45 and IgM were both identified as targets of CD22 in vitro. But there was no evidence that CD45 and IgM were the actual targets physiologically.

Taking a different approach, Paulson and his colleagues decided to settle the question by looking at the target of CD22 in a natural or "in situ" setting. As it turns out, the in vitro experiments that identified CD45 and IgM as candidates for recognition by CD22 were wrong.

Paulson and his colleagues designed a way to attach a common type of cross-linking agent known as a "photoaffinity label" to the sialic acid that would covalently attach to whatever protein it was close to when an ultraviolet light was shined on it. Then they fed the cells the modified form of the sialic acid so that all the glycoproteins on the outside of the cell incorporated this sugar.

This way, when they flipped on the UV light, the CD22 would become permanently attached to whatever glycoprotein to which it was in close proximity—essentially, to the protein to which it was bound. Then, by separating out the components of the cell, they would be able to tell what that molecule was.

What they found was unexpected. CD22 seems to target itself. Neighboring molecules of CD22 target neighboring molecules of CD22, and when they do, they form homo-multimeric complexes.

While there is still much more to understand about how this regulates the activation of the B cell, it is significant because sialic acid is linked to glycoproteins in many ways, and each linkage is unique to the enzyme that makes the sialic acid. B cells have a single gene that makes the sequence that is recognized by CD22, which makes this a good potential target for developing inhibitors that might help to modulate the immune response. 

This work is also significant, says Paulson, because the team's methodprovides a general approach useful for probing what proteins of interest are interacting on the surfaces of living cells and controlling cell signaling.

The article, "Homo-multimeric Complexes of CD22 in B cells Revealed by Protein-Glycan Crosslinking" by Shoufa Han, Brian E. Collins, Per Bengtson, and James C. Paulson will appear in an upcoming issue of the journal Nature Chemical Biology can be accessed at: http://dx.doi.org/10.1038/nchembio713.

This work was supported by the National Institutes of Health and through a fellowship by the Wenner-Gren Foundation.

Send comments to: jasonb@scripps.edu