clusterin  (BioVendor Instruments)

Journal: bioRxiv

Article Title: CD33-CD45 Interaction Reveals a Mechanistic Link to Alzheimer’s Disease Susceptibility

doi: 10.1101/2025.07.28.667311

Figure Lengend Snippet: a. The mass spectrometry findings of the CD33 and CD45 interaction were validated in THP-1 cells using the proximity ligation assay (PLA). Knock-out (KO) of CD33 from the THP-1 cell line led to the elimination of the interaction between CD33 and CD45, as detected by PLA. b. Combining THP-1 cells with either CD33 KO or CD45 KO via CRISPR/Cas9 leads to a reduction in the interaction between CD33 and CD45, suggesting that the interaction is occurring in cis. c. In a solid phase binding assay, recombinant human CD33 bound to immobilized recombinant human CD45 in a dose dependent manner. Increasing concentrations of recombinant hCD33-Fc (0.005µg/mL-5µg/mL) were incubated overnight at 4°C to recombinant hCD45 coated plates and an anti-human Fc antibody was used to detect recombinant bound hCD33. Each dot represents the average of three independent experiments. Two-way ANOVA with Tukey’s multiple comparison test, p*<0.05, p**<0.01, p***<0.001. Asterisks denote comparison of 0.5 μg/mL (blue asterisks) or 1 μg/mL (black asterisk) to 0 μg/mL CD45 concentration treatment. d. Representative images of CD33-CD45 PLA signal in CD33 mutant THP-1 cell lines. Mutations in the sialic acid binding domain (R119K) and the ITIM (Y340F) disrupt the interaction between CD33 and CD45 in THP-1 monocytes, as demonstrated by reduced PLA signal. Mutation in the ITIM-like domain (Y358F) leads to a reduction in CD33-CD45 interaction, as does the double mutant (Y358F/Y340F). e. Quantification of the CD33-CD45 PLA signal in each mutant THP-1 line, as PLA intensity per DAPI positive cell. Each dot represents a technical replicate. Unpaired t-test. *p<0.05, **p<0.01. f. Median fluorescence intensity of CD33-CD45 Flow Cytometry PLA in primary monocytes from 10 individuals, with and without pretreatment with 60nM clusterin (CLU) for 15 minutes. Error bars = mean + SEM. ***p<0.001, paired t-test. g. Representative overlay flow cytometry histogram of CD33-CD45 PLA fluorescent signal from monocytes with and without pretreatment with 60nM clusterin for 15 minutes. h. Median fluorescence intensity of CD33-CD45 PLA in primary monocytes from individuals genotyped for CD33 rs3865444 . N = 16 rs3865444 AA and 18 rs3865444 CC . Error bars = mean + SEM. *p<0.05, unpaired t-test. i. Representative overlayed flow cytometry histogram of PLA fluorescent signal from one CD33 rs3865444 AA and one CD33 rs3865444 CC individual.

Article Snippet: 0.1mg of clusterin (BioVendor, Clusterin Human Native Protein, Human Plasma; Cat. No. 50-206-9196) was resuspended in 108μl of sterile water to make a 12μM stock.

Techniques: Mass Spectrometry, Proximity Ligation Assay, Knock-Out, CRISPR, Binding Assay, Recombinant, Incubation, Comparison, Concentration Assay, Mutagenesis, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: CD33 and clusterin interact biophysically and genetically to modulate Alzheimer risk

doi: 10.1101/2025.07.29.667318

Figure Lengend Snippet: A. Binding curves of clusterin/Aβ42 mixture (left panel) or Aβ42 (right panel). Each binding curve is the average of four independent measurements. Clusterin and Aβ42 concentrations are indicated. B. Steady-state BLI analysis of the clusterin/Aβ42 mixture (red square) or Aβ42 (blue diamond) binding to CD33 M ECD. Error bars represent SD. C. The bar chart of each data point presented in B. Error bars represent SD. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparison test (n = 4, * p <0.05, ** p <0.01 vs clusterin alone).

Article Snippet: Native human plasma-derived clusterin (BioVendor R&D) was reconstituted in sterile water according to the manufacturer’s instructions. iMGs were treated with 300 nM clusterin or sterile water in complete microglia medium for 24 hours.

Techniques: Binding Assay, Comparison

Journal: bioRxiv

Article Title: CD33 and clusterin interact biophysically and genetically to modulate Alzheimer risk

doi: 10.1101/2025.07.29.667318

Figure Lengend Snippet: a. Human plasma CLU alone, and especially human CLU + Aβ oligomers induce ITIM tyrosine phosphorylation of endogenous CD33 (left panel) and recruitment of endogenous SHP-1 (right panel) in human monocytic U937 cells. Quantification of CD33 phosphotyrosine (pY20 antibody immunoreactive anti-CD33 IP products) and of SHP-1 recruitment (anti-SHP-1 immunoreactive anti-CD33 IP products) using band intensity (representative blots are in . * = p <0.05; *** = p <0.001; NS = not significant; ANOVA with Tukey post-hoc correction; error bars = mean ± SEM; For CD33 M -pTyr, No treatment: 1.00±0.0; Aβ treatment: 1.367±0.4107; CLU treatment: 6.283±1.667; CLU + Aβ treatment: 11.92±1.116; For SHP-1, No treatment: 1.00±0.0; Aβ treatment: 1.447±0.2726; CLU treatment: 2.589±0.0688; CLU + Aβ treatment: 4.362±0.5971, n ≥ 3 independent biological replications. b. Recombinant human clusterin (rCLU) and native human plasma clusterin (pCLU), but not desialylated clusterin (desCLU) equivalently activate CD33 ITIM phosphorylation and SHP-1 recruitment in HEK293 cells . CD33 tyrosine phosphorylation and SHP-1 recruitment were measured by western blots band intensity ratios for pY20-phosphotyrosine immunoreactive CD33 versus total CD33 by co-IP western blots. with different cell lines are available in . c. CLU + Aβ induces greater CD33 ITIM phosphorylation than CLU alone, but only in CD33M expressing cells. Quantification of western blots of anti-CD33 immunoprecipitates probed with pY20 anti-phosphotyrosine or anti-HA antibodies reveals that treatment with CLU alone, and especially treatment with CLU + Aβ oligomers, increases CD33 tyrosine phosphorylation in cells expressing either CD33M only or CD33 M plus CD33 m . No CD33 phosphorylation was observed in cells expressing only CD33 m , and very little in CD33 M-5X cells. n = 3 replications. Representative western blots are in . d. CLU + Aβ induces greater CD33 SHP-1 recruitment than CLU alone, but only in CD33M expressing cells. Quantification of western blots of anti-CD33 immunoprecipitates probed with SHP-1 or anti-HA antibodies reveals that treatment with CLU alone, and especially treatment with CLU + Aβ oligomers, increases SHP-1 recruitment in cells expressing either CD33M only or CD33 M plus CD33 m . No SHP-1 recruitment was observed in cells expressing only CD33 m , and very little in CD33 M-5X cells. n = 3 replications. Representative western blots are in . e. Treatment of human monocytes with 0.06 µM CLU for 15 min at 37°C increases binding of CD33 and SHP-1. Human monocytes (genotype CD33 rs3865444 CC ) were treated with CLU and then a proximity ligation assay (PLA) was performed to examine CD33-SHP-1 interaction. Median fluorescence intensity of the PLA of CD33-SHP-1 was measured and analyzed in relation to untreated monocytes from the same individuals. Data were analyzed using paired t-test on GraphPad Prism. *, p<0.05. f. CD33 rs3865444 CC risk genotype-dependent effect of CLUon monomeric Aβ1-42 uptake by human monocytes. Human monocytes (genotype CD33 rs3865444 CC ) were treated with either CLU or desCLU, and the effect on Aβ1-42 uptake was assessed by flow cytometry. CLU significantly reduces the Aβ 1-42 uptake compared to the media control or desCLU treatment. Data were analyzed using geometric mean intensity of HiLyte™ Fluor 647 conjugated Aβ 1-42, n = 10 unrelated donors each examined once. Each dot represents an individual donor. Data were analyzed using ANOVA on GraphPad Prism. **, p<0.01; *, p<0.05.

Article Snippet: Native human plasma-derived clusterin (BioVendor R&D) was reconstituted in sterile water according to the manufacturer’s instructions. iMGs were treated with 300 nM clusterin or sterile water in complete microglia medium for 24 hours.

Techniques: Clinical Proteomics, Phospho-proteomics, Recombinant, Western Blot, Co-Immunoprecipitation Assay, Expressing, Binding Assay, Proximity Ligation Assay, Fluorescence, Flow Cytometry, Control

Journal: bioRxiv

Article Title: CD33 and clusterin interact biophysically and genetically to modulate Alzheimer risk

doi: 10.1101/2025.07.29.667318

Figure Lengend Snippet: Representative western blot of n = 3-4 independent biological replications with different cell lines for . Recombinant human clusterin (rCLU) and native human plasma clusterin (pCLU), but not desialylated clusterin equivalently activate CD33M ITIM phosphorylation and SHP-1 recruitment in HEK293 cells. CD33M tyrosine phosphorylation (top panel) and SHP-1 recruitment (middle panel) were measured by Western blots band intensity ratios for pY20-phosphotyrosine immunoreactive CD33M versus total CD33M (bottom panel) by co-IP western blots..

Article Snippet: Native human plasma-derived clusterin (BioVendor R&D) was reconstituted in sterile water according to the manufacturer’s instructions. iMGs were treated with 300 nM clusterin or sterile water in complete microglia medium for 24 hours.

Techniques: Western Blot, Recombinant, Clinical Proteomics, Phospho-proteomics, Co-Immunoprecipitation Assay