A collaborative effort between two research groups at the Department of Molecular Biology and Genetics, Aarhus University, has led to a breakthrough in our understanding of the membrane-bound protein CD109 – a previously understudied member of the α-macroglobulin family. 

CD109 is a distant relative of proteins such as α2-macroglobulin and complement factor C3, which play central roles in the body's immune defenses. Unlike most members of the family, which circulate freely in the blood, CD109 is anchored to the surface of cells, including activated T cells, keratinocytes, and platelets. It has been shown to affect TGF-β signaling and is believed to be an evolutionary ancestor to both protease inhibitors and complement components.

How CD109 traps proteases

Using biochemical and structural methods, the researchers demonstrated that CD109 acts as a protease inhibitor. Like its relatives, it uses a reactive thioester group that becomes exposed upon cleavage by a protease. This conformational change enables CD109 to form a covalent bond with the protease, effectively neutralizing it.

However, determining how this mechanism works at the molecular level required high-resolution structural insights – which proved to be technically challenging.

Solving a Cryo-EM challenge

PhD students Martin Jørgensen and Kathrine Jensen prepared CD109 samples for cryo-electron microscopy (cryo-EM), but encountered a major obstacle: the protein consistently adopted the same orientation on the EM grid, making structural analysis impossible.

A turning point came when postdoc Ana Almeida joined the project and systematically tested different approaches. She discovered that the detergent CHAPS could overcome the problem, allowing the team to resolve CD109’s structure both before and after protease activation. This revealed a surprising similarity to another well-known protease inhibitor.

Sugar chains that strengthen inhibition

Another striking observation was the presence of glycans surrounding CD109 near the protease-binding site. Ana hypothesized that these sugar chains might act as a molecular shield, preventing proteases from escaping and enhancing the inhibitory effect.

“I needed experimental proof,” Ana explains. “So I used lab-generated enzymes to remove the glycans from CD109 and then compared how well the deglycosylated versus glycosylated forms could inhibit proteases. The result confirmed that the glycans actually contribute to inhibition. That was an exciting moment, because it showed how structural studies can lead to new hypotheses and uncover key mechanistic details.”

Still much to learn

Although many questions remain – such as how CD109’s role in protease inhibition relates to its function in TGF-β signaling – the new structural and functional insights mark a major step forward in understanding this overlooked component of the immune system.

The work was supported by a grant from Independent Research Fund Denmark to Jan J. Enghild.
The article is available open access here: https://www.cell.com/cell-reports/fulltext/S2211-1247(25)00558-3