Body size and digestive system shape resource selection by ungulates : a cross-taxa test of the forage maturation hypothesis

The forage maturation hypothesis (FMH) states that energy intake for ungulates is maximised when forage biomass is at intermediate levels. Nevertheless, metabolic allometry and different digestive systems suggest that resource selection should vary across ungulate species. By combining GPS relocations with remotely sensed data on forage characteristics and surface water, we quantified the effect of body size and digestive system in determining movements of 30 populations of hindgut fermenters (equids) and ruminants across biomes. Selection for intermediate forage biomass was negatively related to body size, regardless of digestive system. Selection for proximity to surface water was stronger for equids relative to ruminants, regardless of body size. To be more generalisable, we suggest that the FMH explicitly incorporate contingencies in body size and digestive system, with small-bodied ruminants selecting more strongly for potential energy intake, and hindgut fermenters selecting more strongly for surface water.DATA AVAILABILITY STATEMENT : The dataset used in our analyses is available via Dryad repository (https://doi.org/10.5061/dryad.jsxksn09f) following a year-long embargo from publication of the manuscript. The coordinates associated with mountain zebra data are not provided in an effort to protect critically endangered black rhino (Diceros bicornis) locations. Interested researchers can contact the data owner (Minnesota Zoo) directly for inquiries.https://wileyonlinelibrary.com/journal/elehj2022Mammal Research InstituteZoology and Entomolog

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin–ankyrin G119 skeleton in Madin Darby canine kidney cells

Spectrin (βIΣ∗) and ankyrin (Ank(G119)) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of βI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of α- and β-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of β-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as Ank(G119), Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular–tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin–ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins

Ankyrin facilitates intracellular trafficking of α1-Na+-K+-ATPase in polarized cells

Defects in ankyrin underlie many hereditary disorders involving the mislocalization of membrane proteins. Such phenotypes are usually attributed to ankyrin's role in stabilizing a plasma membrane scaffold, but this assumption may not be accurate. We found in Madin-Darby canine kidney cells and in other cultured cells that the 25-residue ankyrin-binding sequence of α1-Na+-K+-ATPase facilitates the entry of α1,β1-Na+-K+-ATPase into the secretory pathway and that replacement of the cytoplasmic domain of vesicular stomatitis virus G protein (VSV-G) with this ankyrin-binding sequence bestows ankyrin dependency on the endoplasmic reticulum (ER) to Golgi trafficking of VSV-G. Expression of the ankyrin-binding sequence of α1-Na+-K+-ATPase alone as a soluble cytosolic peptide acts in trans to selectively block ER to Golgi transport of both wild-type α1-Na+-K+-ATPase and a VSV-G fusion protein that includes the ankyrin-binding sequence, whereas the trafficking of other proteins remains unaffected. Similar phenotypes are also generated by small hairpin RNA-mediated knockdown of ankyrin R or the depletion of ankyrin in semipermeabilized cells. These data indicate that the adapter protein ankyrin acts not only at the plasma membrane but also early in the secretory pathway to facilitate the intracellular trafficking of α1-Na+-K+-ATPase and presumably other selected proteins. This novel ankyrin-dependent assembly pathway suggests a mechanism whereby hereditary disorders of ankyrin may be manifested as diseases of membrane protein ER retention or mislocalization

A widely expressed βIII spectrin associated with Golgi and cytoplasmic vesicles

Spectrin is an important structural component of the plasma membrane skeleton. Heretofore-unidentified isoforms of spectrin also associate with Golgi and other organelles. We have discovered another member of the β-spectrin gene family by homology searches of the GenBank databases and by 5′ rapid amplification of cDNA ends of human brain cDNAs. Collectively, 7,938 nucleotides of contiguous clones are predicted to encode a 271,294-Da protein, called βIII spectrin, with conserved actin-, protein 4.1-, and ankyrin-binding domains, membrane association domains 1 and 2, a spectrin dimer self-association site, and a pleckstrin-homology domain. βIII spectrin transcripts are concentrated in the brain and present in the kidneys, liver, and testes and the prostate, pituitary, adrenal, and salivary glands. All of the tested tissues contain major 9.0-kb and minor 11.3-kb transcripts. The human βIII spectrin gene (SPTBN2) maps to chromosome 11q13 and the mouse gene (Spnb3) maps to a syntenic region close to the centromere on chromosome 19. Indirect immunofluorescence studies of cultured cells using antisera specific to human βIII spectrin reveal a Golgi-associated and punctate cytoplasmic vesicle-like distribution, suggesting that βIII spectrin associates with intracellular organelles. This distribution overlaps that of several Golgi and vesicle markers, including mannosidase II, p58, trans-Golgi network (TGN)38, and β-COP and is distinct from the endoplasmic reticulum markers calnexin and Bip. Liver Golgi membranes and other vesicular compartment markers cosediment in vitro with βIII spectrin. βIII spectrin thus constitutes a major component of the Golgi and vesicular membrane skeletons

ADP ribosylation factor regulates spectrin binding to the Golgi complex

Homologues of two major components of the well-characterized erythrocyte plasma-membrane-skeleton, spectrin (a not-yet-cloned isoform, βIΣ* spectrin) and ankyrin (Ank(G119) and an ≈195-kDa ankyrin), associate with the Golgi complex. ADP ribosylation factor (ARF) is a small G protein that controls the architecture and dynamics of the Golgi by mechanisms that remain incompletely understood. We find that activated ARF stimulates the in vitro association of βIΣ* spectrin with a Golgi fraction, that the Golgi-associated βIΣ* spectrin contains epitopes characteristic of the βIΣ2 spectrin pleckstrin homology (PH) domain known to bind phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), and that ARF recruits βIΣ* spectrin by inducing increased PtdInsP(2) levels in the Golgi. The stimulation of spectrin binding by ARF is independent of its ability to stimulate phospholipase D or to recruit coat proteins (COP)-I and can be blocked by agents that sequester PtdInsP(2). We postulate that a PH domain within βIΣ* Golgi spectrin binds PtdInsP(2) and acts as a regulated docking site for spectrin on the Golgi. Agents that block the binding of spectrin to the Golgi, either by blocking the PH domain interaction or a constitutive Golgi binding site within spectrin’s membrane association domain I, inhibit the transport of vesicular stomatitis virus G protein from endoplasmic reticulum to the medial compartment of the Golgi complex. Collectively, these results suggest that the Golgi-spectrin skeleton plays a central role in regulating the structure and function of this organelle

Improving the Pharmacodynamics and In Vivo Activity of ENPP1‐Fc Through Protein and Glycosylation Engineering

Enzyme replacement with ectonucleotide pyrophosphatase phospodiesterase‐1 (ENPP1) eliminates mortality in a murine model of the lethal calcification disorder generalized arterial calcification of infancy. We used protein engineering, glycan optimization, and a novel biomanufacturing platform to enhance potency by using a three‐prong strategy. First, we added new N‐glycans to ENPP1; second, we optimized pH‐dependent cellular recycling by protein engineering of the Fc neonatal receptor; finally, we used a two‐step process to improve sialylation by first producing ENPP1‐Fc in cells stably transfected with human α‐2,6‐sialyltransferase (ST6) and further enhanced terminal sialylation by supplementing production with 1,3,4‐ O ‐Bu 3 ManNAc. These steps sequentially increased the half‐life of the parent compound in rodents from 37 hours to ~ 67 hours with an added N‐glycan, to ~ 96 hours with optimized pH‐dependent Fc recycling, to ~ 204 hours when the therapeutic was produced in ST6‐overexpressing cells with 1,3,4‐ O ‐Bu 3 ManNAc supplementation. The alterations were demonstrated to increase drug potency by maintaining efficacious levels of plasma phosphoanhydride pyrophosphate in ENPP1‐deficient mice when the optimized biologic was administered at a 10‐fold lower mass dose less frequently than the parent compound—once every 10 days vs. 3 times a week. We believe these improvements represent a general strategy to rationally optimize protein therapeutics