A Human IgSF Cell-Surface Interactome Reveals a Complex Network of Protein-Protein Interactions.

Cell-surface protein-protein interactions (PPIs) mediate cell-cell communication, recognition, and responses. We executed an interactome screen of 564 human cell-surface and secreted proteins, most of which are immunoglobulin superfamily (IgSF) proteins, using a high-throughput, automated ELISA-based screening platform employing a pooled-protein strategy to test all 318,096 PPI combinations. Screen results, augmented by phylogenetic homology analysis, revealed ∼380 previously unreported PPIs. We validated a subset using surface plasmon resonance and cell binding assays. Observed PPIs reveal a large and complex network of interactions both within and across biological systems. We identified new PPIs for receptors with well-characterized ligands and binding partners for "orphan" receptors. New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous system development and function, differentiation/proliferation, metabolism, vascularization, and reproduction. These PPIs provide a resource for further biological investigation into their functional relevance and may offer new therapeutic drug targets.

Guide Swap enables genome-scale pooled CRISPR-Cas9 screening in human primary cells.

CRISPR-Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells-an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR-Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4+ T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34+ hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.

Optimization of an Enzymatic Antibody-Drug Conjugation Approach Based on Coenzyme A Analogs.

Phosphopantetheine transferases (PPTases) can be used to efficiently prepare site-specific antibody-drug conjugates (ADCs) by enzymatically coupling coenzyme A (CoA)-linker payloads to 11-12 amino acid peptide substrates inserted into antibodies. Here, a two-step strategy is established wherein in a first step, CoA analogs with various bioorthogonal reactivities are enzymatically installed on the antibody for chemical conjugation with a cytotoxic payload in a second step. Because of the high structural similarity of these CoA analogs to the natural PPTase substrate CoA-SH, the first step proceeds very efficiently and enables the use of peptide tags as short as 6 amino acids compared to the 11-12 amino acids required for efficient one-step coupling of the payload molecule. Furthermore, two-step conjugation provides access to diverse linker chemistries and spacers of varying lengths. The potency of the ADCs was largely independent of linker architecture. In mice, proteolytic cleavage was observed for some C-terminally linked auristatin payloads. The in vivo stability of these ADCs was significantly improved by reduction of the linker length. In addition, linker stability was found to be modulated by attachment site, and this, together with linker length, provides an opportunity for maximizing ADC stability without sacrificing potency.

Efficient Preparation of Site-Specific Antibody-Drug Conjugates Using Phosphopantetheinyl Transferases.

Post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases) has previously been used to site-specifically label proteins with structurally diverse molecules. PPTase catalysis results in covalent modification of a serine residue in acyl/peptidyl carrier proteins and their surrogate substrates which are typically fused to the N- or C-terminus. To test the utility of PPTases for preparing antibody-drug conjugates (ADCs), we inserted 11 and 12-mer PPTase substrate sequences at 110 constant region loop positions of trastuzumab. Using Sfp-PPTase, 63 sites could be efficiently labeled with an auristatin toxin, resulting in 95 homogeneous ADCs. ADCs labeled in the CH1 domain displayed in general excellent pharmacokinetic profiles and negligible drug loss. A subset of CH2 domain conjugates underwent rapid clearance in mouse pharmacokinetic studies. Rapid clearance correlated with lower thermal stability of the particular antibodies. Independent of conjugation site, almost all ADCs exhibited subnanomolar in vitro cytotoxicity against HER2-positive cell lines. One selected ADC was shown to induce tumor regression in a xenograft model at a single dose of 3 mg/kg, demonstrating that PPTase-mediated conjugation is suitable for the production of highly efficacious and homogeneous ADCs.

Structure and function of the DUF2233 domain in bacteria and in the human mannose 6-phosphate uncovering enzyme.

DUF2233, a domain of unknown function (DUF), is present in many bacterial and several viral proteins and was also identified in the mammalian transmembrane glycoprotein N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase ("uncovering enzyme" (UCE)). We report the crystal structure of BACOVA_00430, a 315-residue protein from the human gut bacterium Bacteroides ovatus that is the first structural representative of the DUF2233 protein family. A notable feature of this structure is the presence of a surface cavity that is populated by residues that are highly conserved across the entire family. The crystal structure was used to model the luminal portion of human UCE (hUCE), which is involved in targeting of lysosomal enzymes. Mutational analysis of several residues in a highly conserved surface cavity of hUCE revealed that they are essential for function. The bacterial enzyme (BACOVA_00430) has ∼1% of the catalytic activity of hUCE toward the substrate GlcNAc-P-mannose, the precursor of the Man-6-P lysosomal targeting signal. GlcNAc-1-P is a poor substrate for both enzymes. We conclude that, for at least a subset of proteins in this family, DUF2233 functions as a phosphodiester glycosidase.