Biology subjects

Metzendorf, N. G.

Publications and source records attributed to Metzendorf, N. G..

3 recordsLinked to original sources

bioRxiv2026

Paralemmin-2 is a membrane-anchored cytoskeletal constituent of Axon Initial Segments and nodes of Ranvier

The axon initial segment (AIS) and nodes of Ranvier (NoR) are essential for action potential initiation and propagation. They share many features of their molecular architecture, and their assembly mechanisms converge on the membrane-associated periodic skeleton (MPS). Here, we identify Paralemmin-2 (Palm2) as a component of both the AIS and NoR. Palm2 depletion shortens the AIS and reduces neuronal excitability. Endogenous Palm2 in the AIS is non-periodic, but upon overexpression it associates with the MPS, localizing to actin rings and reducing {beta}IV-spectrin abundance and periodicity. In NoR of the central and peripheral nervous system, Palm2 localizes to different subdomains - nodal or paranodal, respectively. Palm2, and its homolog Palm1, bind the deubiquitinase USP7, implicating paralemmins in proteostasis at the MPS. The complementary localizations of Palm2 and Palm1 at the AIS/NoR or axon shafts, respectively, parallel the distributions of {beta}-spectrin and ankyrin isoforms between these axonal compartments. We propose that Palm2 modulates the submembrane cytoskeleton and its membrane attachment, and thus contributes to the assembly, functioning and remodeling of the AIS and NoR.

cell biology↗

bioRxiv2025

Engineered Fab glycosylation of a blood-brain barrier transporter single-chain variable fragment preserved functionality but did not impact anti-drug antibody formation

The single-chain variable fragment (scFv) of the 8D3 antibody enables blood-brain barrier transport via transferrin receptor-mediated transcytosis. However, when fused to therapeutic antibodies, scFv8D3 increased immunogenicity and triggered anti-drug antibodies (ADA). Sialylation of N-linked glycans can critically influence protein immunogenicity, potentially conferring immunosuppressive properties and extended half-life. We investigated whether introducing sialic acid residues to scFv8D3 through engineered fragment antigen-binding (Fab) glycosylation could reduce ADA formation. We generated two novel variants with distinct N-linked glycosylation motifs (NXT/S), which both displayed sialic acid residues and retained functionality, as evidenced by the preservation of transferrin receptor binding and brain uptake. However, Fab glycosylation did not significantly alter pharmacokinetics or ADA production in mice. Our findings demonstrated that introducing Fab glycosylation is feasible, without compromising biological activity. Nonetheless, this strategy alone was insufficient to mitigate immunogenicity, underscoring the need for alternative approaches to improve the long-term safety of brain-targeting therapeutics. Statement of significanceSialylation of N-linked glycans can critically influence protein immunogenicity. We introduced N-linked Fab glycosylation sites into a scFv-based therapeutic construct with the aim of reducing ADA production upon repeated administration to mice. Although the sialylated variants retained functionality, pharmacokinetics and ADA levels remained unaffected, suggesting limited immunomodulation via engineered sialylation.

bioengineering↗

bioRxiv2024

Paralemmin-1 controls the nanoarchitecture of the neuronal submembrane cytoskeleton

The Membrane-associated Periodic Skeleton (MPS) is a specialized submembrane cytoskeleton of neuronal cells, characterized by a highly ordered 190 nm periodic lattice, with emerging functions in mechanical resilience, inter- and intracellular signaling, and action potential transmission. Here, we identify Paralemmin-1 (Palm1) as a new component and regulator of the MPS. Palm1 binds to the N-terminal region of {beta}II-spectrin, a core MPS component, and is periodically organized along the axon in hippocampal neurons. Applying the 3D imaging power of MINFLUX, we locate Palm1 in close proximity (<20 nm) to the actin-capping protein and MPS component adducin. Functionally, Palm1 overexpression enhances the degree of periodicity of several MPS proteins ({beta}II-spectrin, adducin, and ankyrinB) without altering their local concentrations, while the knock-out severely compromises the MPS structure and modifies electrophysiological properties of neurons. Both the MPS-binding and remodelling activities of Palm1 are abolished by mutating a single amino acid (W54A) in the conserved Paralemmin sequence motif. Our findings identify Palm1 as the first protein specifically dedicated to organizing the MPS, and will advance the understanding of the regulation of MPS assembly and remodelling, as well as of the Paralemmin protein family.

cell biology↗