Alex Guseman

Engineered OAA lectins as selective and sensitive high mannose glycan targeting tools https://www.biorxiv.org/content/10.64898/2026.03.04.709641v1

This was spearheaded by a wonderful postdoc in the lab Bryce Ackermann with a great supporting cast. I hope yall give it a read and we would be happy to take suggestions on where to send it (or if you are an editor and want it in your journal let me know). #biophysics #glycotime #proteinevolution

To Summarize we used phage display to identify variants with enhanced glycan specificity and enhanced affinity. We characterized these structurally and biophysically and demonstrated their use as tools for glycoform specific biology or for increased antiviral effects.

As OAA was originally discovered as an antiviral and previously I showed it can inhibit SARS-CoV-2 Viral entry, demonstrated that V4V4 and PM6PM6 bivalent OAAs have differential neutralization properties, as V4V4 losses the ability to block viral entry, and PM6PM6 shows a 4x better inhibitor.

To look at the ability of V4 and PM6 to interact with specific glycoforms of proteins we turned to RNAse B which has a single glycosylation site and that can be Man5/6/7/8/9. We used BLI and pulldowns to show that V4 interacts only with the M5 glycoform, while PM6 interacts with all glycoforms

More impressively bivalent V4V4 tuned its selectivity, showing Man-5 binding at 18 nM but Man-6 and Man-7 binidng at 3 and 13 uM, representing a 166x (Man6) and 722x (Man7) fold more selective for Man 5. Based off of this we wanted to demonstrate the use of V4 as a tool in biological systems.

As OAA is symetric with two identical binding sites (which we knocked one out for phage dispaly). We wanted to know if these properties can be tuned by re-installing multivalency. Our bivalent PM6PM6 showed a 26x enhancement in Man-9 binding compared to the bivalent WT!

We believe that this affinity gain offsets the loss in affinity caused by the mutations that confer selectivity. Thus discovering this variant was likely only achievable in a combinatorial library screen. We also noticed that this variant PM6 has enhanced glycan binding!

In order to dissect which mutation(s) conferred which property we used a set of 25 point mutations and BLI. Our data allowed us to bind variants into affinity enhancing, specificity enhancing, weakening or destructive bins. Uniquely we found the two tyrosine mutants cause a increase to Kd!

We solved the crystal structure of bivalent V4V4 bound to the Man-5 core glycan. this revealed a series of interactions that caused a loop shift and the creation of a steric hindrance where the next sugar of a Man-6 glycan would bind. Thus providing a structural basis for specificity.

We screened each variant of interest for binding to HMGs using BLI. From this two variants of interest jumped out right away V4, showing Man-5 specificity, and V6 showing specificity for just Man-5 and Man-6. Showing specificity for a single Glycoform, we further investigated V4.

We fished our library for variants with enhanced binding to Man-5 and over four rounds we observed selection across the loop regions of interest. Using a bioinformatics pipeline we subsequently identified sequences of interest, and cloned them for expression and purification.

OAA is a beta barrel protein that binds glycans in its loop regions, it is incredibly structurally stable, and we can control its valency by mutation. Thus we used it as scaffold to make a 5 x 10*8 variant library of a single carbohydrate recognition domain for phage display.

Glycosylation is a heterogenous process, as such many lectins that bind one glycan, bind similar glycan structures. A great example of this is the OAA lectin that binds high mannose glycans. It recognizes the Man-5 core structure present in all high mannose glycans (HMGs, Man-5/6/7/8/9).

I am REALLY excited to share this manuscript from my group now on bioarxiv. Here we used the OAA lectin scaffold to ask can lectins be evolved to have specific interactions for defined glycan structures . www.biorxiv.org/content/10.6...

I need my those bioarxiv manuscript vetters to vet my manuscript asap because i cant wait to share it with yall...

Nuclear Magnetic Resonance Facility Lead (NMR Facility Manager) the Department of Chemistry and Biochemistry, California State University, San Diego, CA careers.sdsu.edu/en-us/job/55... #NMRchat #NMR #NMRjobs 🧲

quick call pam in to testify again!

You know I thought they have been rock solid for like ~50 years on average...

If needed You can do a CBP tariff exemption form. I just had to do one for a bruker purchase that wanted another 100k in tariffs.

I feel like now will be a rush to get the rest of the remaining 4 letter PDB codes...

In a couple of weeks we will be dropping a preprint (w/ one of the coolest PDB codes btw...) so stay tuned...

it would be great if each journal had a list what each status for their "manuscript status" types mean somewhere.... and the NIH too...

Me: ordering Girl Scout cookies online feels so wrong

Also me: oh (grad students kid) has a link to order send it my way…

The number of folks that have still asked for letters despite me answering this way is still surprisingly high…

living in san diego this is a real fear of mine. It was a high of 75 yesterday, if i couldnt have gotten my afternoon ice coffee while on my daily beach walk in between meetings how could I do my job?

Did I miss something? I have been using it for like 5 years and never had an issue. I sat this cycle out but did they change it ?

Better but still not the Fetterman who Pa voted for.

i had a not great score that ended up getting picked up. dont lose hope.

have you talked to the PO yet?

another day another delta lounge?

I’d argue that model proteins already exist and those of us who work with them (e.g. gb1 or ubiquitin) to benchmark new biophysics constantly get criticized for how relevant these studies are and being told to do it on a real protein.