The apical sorting signal on human aminopeptidase N is not located in the stalk but in the catalytic head group

Abstract ADAMTS13, a member of a disintegrin and metalloprotease with thrombospondin type 1 repeats family, is mainly synthesized in the liver and secreted into the blood stream. It limits platelet aggregation in small arteries by cleaving the Tyr1605-Met1606 bond of von Willebrand factor (VWF). ADAMTS13 consists of a propeptide, a metalloprotease domain and a disintegrin domain, first thrombospondin type 1 repeat (TSP1), followed by a Cys-rich domain and a spacer domain. The carboxyl terminus of ADAMTS13 contains seven more TSP1 repeats and two CUB domains. The distal carboxyl terminal TSP1 repeats and CUB domains of ADAMTS13 appeared not to be essential for cleavage of VWF, but they might be important for ADAMTS13 biosynthesis and intracellular sorting. In this study, we determined: 1) the polarity of ADAMTS13 secretion in MDCK cells; 2) the signals that direct polarized sorting of ADAMTS13; 3) the platform by which ADAMTS13 is sorted. MDCK II cells were stably transfected with plasmids encoding a full-length ADAMTS13 and a series of carboxyl termini-deleted ADAMTS13 variants. The polarity of full-length ADAMTS13 and its variants was determined in a Transwell™ filter system by Western blotting with anti-V5 and by proteolytic activity toward VWF in the conditioned medium. We showed that full-length ADAMTS13 was predominantly sorted apically, with an approximately 75% of protein and 85% of proteolytic activity of ADAMTS13 detected in the apical domain of MDCK cells. An ADAMTS13 variant (del1) lacking two CUB domains was randomly sorted into both apical and basolateral domains of MDCK cells, with an approximately 52% of protein and 49% of proteolytic activity detected apically. The ADAMTS13 variant (del2) deleted after the spacer domain was also randomly secreted, but the ADAMTS13 variant (del3) deleted after the Cys-rich domain was sorted apically, with an approximately 92% of protein detected in the apical domain of MDCK cells. These data suggested the presence of an apical sorting signal in the CUB domains and Cys-rich domain, and an inhibitory sorting signal in the spacer domain and/or in the TSP1 2~8 repeats. Moreover, the ADAMTS13 variant (del4) deleted after the first TSP1 was predominantly sorted into the basolateral domain of MDCK cells, with an approximately 30% of protein detected apically, suggesting the presence of basolateral signal in the first TSP1 repeat. The ADAMTS13 variants deleted after the disintegrin domain (del5) and the metalloprotease domain (del6) were sorted apically again. These data suggested the presence of multiple sorting signals in ADAMTS13 protein. The polarity of ADAMTS13 sorting may depend on the overall strength of apical, basolateral and inhibitory signals in the protein. By Triton-X 100 solubility at 4 °C and sucrose gradient analyses, we showed that full-length ADAMTS13 protein was detected in top third and fourth fraction of the gradient where a plasma membrane protein, caveolin-1 was located, suggesting that full-length ADAMTS13 was incorporated into the detergent-insoluble glycosphingolipid and cholesterol-enriched microdomains, i.e lipid rafts in the biosynthetic and trafficking pathway. Deletion of two CUB domains abolished ADAMTS13 association with lipid rafts and randomized its sorting. Therefore, we conclude that apical sorting of ADAMTS13 in MDCK cells may be mediated by lipid rafts, and that the CUB domains of ADAMTS13 are required for raft association and apical sorting of ADAMTS13.

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Tyrosine sulphation is not required for microvillar expression of intestinal aminopeptidase N

Biochemical Journal ◽

10.1042/bj2540219 ◽

1988 ◽

Vol 254 (1) ◽

pp. 219-222 ◽

Cited By ~ 4

Author(s):

E M Danielsen

Keyword(s):

Protein Synthesis ◽

Aminopeptidase N ◽

Sorting Signal ◽

Mucosal Explants

The effect of 2,6-dichloro-4-nitrophenol (DCNP), an inhibitor of phenol sulphotransferases (EC 2.8.2.-), on the biosynthesis of aminopeptidase N (EC 3.4.11.2) was studied in organ-cultured pig intestinal mucosal explants. At 50 microM DCNP did not affect protein synthesis but it decreased incorporation of [35S]sulphate into aminopeptidase N and other major microvillar hydrolases by 70-85% compared with controls, indicating an inhibition of their post-translational tyrosine sulphation. In labelling experiments with [35S]methionine from 0.5 to 5 h, DCNP was tested for its possible influence on synthesis, processing and microvillar expression of aminopeptidase N, but no effect on any of these parameters could be detected. It can therefore be concluded that tyrosine sulphation is not required (for instance as a sorting signal) for the targeting of newly synthesized enzymes to the microvillar membrane.

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A basolateral sorting signal is encoded in the α-subunit of Na-K-ATPase

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.3.c688 ◽

1998 ◽

Vol 274 (3) ◽

pp. C688-C696 ◽

Cited By ~ 37

Author(s):

T. R. Muth ◽

C. J. Gottardi ◽

D. L. Roush ◽

M. J. Caplan

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Surface Expression ◽

Apical Surface ◽

Amino Acid Residues ◽

Β Subunit ◽

Α Subunit ◽

Sorting Signal ◽

Apical Sorting ◽

Gastric Parietal Cells

Na-K-ATPase and H-K-ATPase are highly homologous ion pumps that exhibit distinct plasma membrane distributions in epithelial cells. We have studied the α-subunits of these heterodimeric pumps to identify the protein domains responsible for their polarized sorting. A chimeric α-subunit construct (N519H) was generated in which the first 519 amino acid residues correspond to the Na-K-ATPase sequence and the remaining 500 amino acids are derived from the H-K-ATPase sequence. In stably transfected LLC-PK1cell lines, we found that the N519H chimera is restricted to the basolateral surface under steady-state conditions, suggesting that residues within the NH2-terminal 519 amino acids of the Na-K-ATPase α-subunit contain a basolateral sorting signal. H-K-ATPase β-subunit expressed alone in LLC-PK1cells accumulates at the apical surface. When coexpressed with N519H, the H-K-ATPase β-subunit assembles with this chimera and accompanies it to the basolateral surface. Thus the NH2-terminal basolateral signal in the Na-K-ATPase α-subunit masks or is dominant over any apical sorting information present in the β-polypeptide. In gastric parietal cells, the H-K-ATPase β-subunit targets the H-K-ATPase to an intracellular vesicular compartment which fuses with the plasma membrane in response to secretagogue stimulation. To test whether the chimera-H-K-ATPase β-subunit complex is directed to a similar compartment in LLC-PK1cells, we treated transfected cells with drugs that raise intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) levels. Elevation of cytosolic cAMP increased the surface expression of both the N519H chimera and the H-K-ATPase β-subunit. This increase in surface expression, however, appears to be the result of transcriptional upregulation and not recruitment of chimera to the surface from a cAMP-inducible compartment.

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Exon Loss Accounts for Differential Sorting of Na-K-Cl Cotransporters in Polarized Epithelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e08-05-0478 ◽

2008 ◽

Vol 19 (10) ◽

pp. 4341-4351 ◽

Cited By ~ 59

Author(s):

Monica Carmosino ◽

Ignacio Giménez ◽

Michael Caplan ◽

Biff Forbush

Keyword(s):

Gene Encoding ◽

Renal Epithelial Cell ◽

Sorting Signal ◽

C Terminus ◽

Dileucine Motif ◽

Epithelial Cell Lines ◽

Amino Acid Stretch ◽

Additional Exon ◽

Apical Membranes ◽

Apical Sorting

The renal Na-K-Cl cotransporter (NKCC2) is selectively expressed in the apical membranes of cells of the mammalian kidney, where it is the target of the clinically important loop diuretics. In contrast, the “secretory” NKCC1 cotransporter is localized in the basolateral membranes of many epithelia. To identify the sorting signal(s) that direct trafficking of NKCCs, we generated chimeras between the two isoforms and expressed these constructs in polarized renal epithelial cell lines. This analysis revealed an amino acid stretch in NKCC2 containing apical sorting information. The NKCC1 C terminus contains a dileucine motif that constitutes the smallest essential component of its basolateral sorting signal. NKCC1 lacking this motif behaves as an apical protein. Examination of the NKCC gene structure reveals that this dileucine motif is encoded by an additional exon in NKCC1 absent in NKCC2. Phylogenetic analysis of this exon suggests that the evolutionary loss of this exon from the gene encoding the basolateral NKCC1 constitutes a novel mechanism that accounts for the apical sorting of the protein encoded by the NKCC2 gene.

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Dual Inhibitors of Enkephalin-Degrading Enzymes (Neutral Endopeptidase 24.11 and Aminopeptidase N) as Potential New Medications in the Management of Pain and Opioid Addiction

PsycEXTRA Dataset ◽

10.1037/e495822006-007 ◽

1995 ◽

Cited By ~ 1

Author(s):

Bernard P. Roques ◽

◽

Florence Noble

Keyword(s):

Neutral Endopeptidase ◽

Opioid Addiction ◽

Aminopeptidase N ◽

Degrading Enzymes ◽

Endopeptidase 24.11 ◽

Dual Inhibitors

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Mechanisms of Drug Solubilization by Polar Lipids in Biorelevant Media

10.26434/chemrxiv.12649145.v1 ◽

2020 ◽

Author(s):

Vladimir Katev ◽

Zahari Vinarov ◽

Slavka S. Tcholakova

Keyword(s):

Chain Length ◽

Acyl Chain ◽

Polar Lipids ◽

Superior Performance ◽

Head Group ◽

Formulation Development ◽

Biorelevant Media ◽

Drug Solubilization ◽

Colloidal Aggregates ◽

Class 1

Despite the widespread use of lipid excipients in both academic research and oral formulation development, rational selection guidelines are still missing. In the current study, we aimed to establish a link between the molecular structure of commonly used polar lipids and drug solubilization in biorelevant media. We studied the effect of 26 polar lipids of the fatty acid, phospholipid or monoglyceride type on the solubilization of fenofibrate in a two-stage in vitro GI tract model. The main trends were checked also with progesterone and danazol. Based on their fenofibrate solubilization efficiency, the polar lipids can be grouped in 3 main classes. Class 1 substances (n = 5) provide biggest enhancement of drug solubilization (>10-fold) and are composed only by unsaturated compounds. Class 2 materials (n = 10) have an intermediate effect (3-10 fold increase) and are composed primarily (80 %) of saturated compounds. Class 3 materials (n = 11) have very low or no effect on drug solubilization and are entirely composed of saturated compounds. The observed behaviour of the polar lipids was rationalized by using two classical physicochemical parameters: the acyl chain phase transition temperature (Tm) and the critical micellar concentration (CMC). Hence, the superior performance of class 1 polar lipids was explained by the double bonds in their acyl chains, which: (1) significantly decrease Tm, allowing these C18 lipids to form colloidal aggregates and (2) prevent tight packing of the molecules in the aggregates, resulting in bigger volume available for drug solubilization. Long-chain (C18) saturated polar lipids had no significant effect on drug solubilization because their Tm was much higher than the temperature of the experiment (T = 37 C) and, therefore, their association in colloidal aggregates was limited. On the other end of the spectrum, the short chain octanoic acid manifested a high CMC (50 mM), which had to be exceeded in order to enhance drug solubilization. When these two parameters were satisfied (C > CMC, Tm < Texp), the increase of the polar lipid chain length increased the drug solubilization capacity (similarly to classical surfactants), due to the decreased CMC and bigger volume available for solubilization. The hydrophilic head group also has a dramatic impact on the drug solubilization enhancement, with polar lipids performance decreasing in the order: choline phospholipids > monoglycerides > fatty acids. As both the acyl chain length and the head group type are structural features of the polar lipids, and not of the solubilized drugs, the impact of Tm and CMC on solubilization by polar lipids should hold true for a wide variety of hydrophobic molecules. The obtained mechanistic insights can guide rational drug formulation development and thus support modern drug discovery pipelines.

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Mechanisms of Drug Solubilization by Polar Lipids in Biorelevant Media

10.26434/chemrxiv.12649145 ◽

2020 ◽

Author(s):

Vladimir Katev ◽

Zahari Vinarov ◽

Slavka S. Tcholakova

Keyword(s):

Chain Length ◽

Acyl Chain ◽

Polar Lipids ◽

Superior Performance ◽

Head Group ◽

Formulation Development ◽

Biorelevant Media ◽

Drug Solubilization ◽

Colloidal Aggregates ◽

Class 1

Despite the widespread use of lipid excipients in both academic research and oral formulation development, rational selection guidelines are still missing. In the current study, we aimed to establish a link between the molecular structure of commonly used polar lipids and drug solubilization in biorelevant media. We studied the effect of 26 polar lipids of the fatty acid, phospholipid or monoglyceride type on the solubilization of fenofibrate in a two-stage in vitro GI tract model. The main trends were checked also with progesterone and danazol. Based on their fenofibrate solubilization efficiency, the polar lipids can be grouped in 3 main classes. Class 1 substances (n = 5) provide biggest enhancement of drug solubilization (>10-fold) and are composed only by unsaturated compounds. Class 2 materials (n = 10) have an intermediate effect (3-10 fold increase) and are composed primarily (80 %) of saturated compounds. Class 3 materials (n = 11) have very low or no effect on drug solubilization and are entirely composed of saturated compounds. The observed behaviour of the polar lipids was rationalized by using two classical physicochemical parameters: the acyl chain phase transition temperature (Tm) and the critical micellar concentration (CMC). Hence, the superior performance of class 1 polar lipids was explained by the double bonds in their acyl chains, which: (1) significantly decrease Tm, allowing these C18 lipids to form colloidal aggregates and (2) prevent tight packing of the molecules in the aggregates, resulting in bigger volume available for drug solubilization. Long-chain (C18) saturated polar lipids had no significant effect on drug solubilization because their Tm was much higher than the temperature of the experiment (T = 37 C) and, therefore, their association in colloidal aggregates was limited. On the other end of the spectrum, the short chain octanoic acid manifested a high CMC (50 mM), which had to be exceeded in order to enhance drug solubilization. When these two parameters were satisfied (C > CMC, Tm < Texp), the increase of the polar lipid chain length increased the drug solubilization capacity (similarly to classical surfactants), due to the decreased CMC and bigger volume available for solubilization. The hydrophilic head group also has a dramatic impact on the drug solubilization enhancement, with polar lipids performance decreasing in the order: choline phospholipids > monoglycerides > fatty acids. As both the acyl chain length and the head group type are structural features of the polar lipids, and not of the solubilized drugs, the impact of Tm and CMC on solubilization by polar lipids should hold true for a wide variety of hydrophobic molecules. The obtained mechanistic insights can guide rational drug formulation development and thus support modern drug discovery pipelines.

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