Subunit structure of wheat germ agglutinin

Cells isolated by enzymic digestion of embryonic tendon were incubated under N₂ so that they synthesized and accumulated the unhydroxylated form of procollagen which is known as protocollagen and which is largely comprised of pro-α chains linked by interchain disulfide bonds. The cells were then exposed to O₂ so that the intracellular protocollagen was hydroxylated and secreted as procollagen. When the hydroxylation was allowed to proceed at 31° or 34°, the procollagen secreted into the medium was triple-helical but its hydroxyproline content was less than two-thirds and its hydroxylysine content was less than half the control. Even when the hydroxylation was allowed to occur at 37°, the procollagen secreted by the cells was under-hydroxylated by about 15% in terms of its hydroxyproline content and about 45% in terms of its hydroxylysine content. The results may have consequences for collagen synthesis by tendons and similar tissues $\text{in vivo}$, since temporary anoxia in such tissues may well lead to the synthesis of a less stable procollagen or to fibers of decreased tensile strength.     

Purification and partial characterization of the type II procollagen synthesized by embryonic cartilage cells

Matrix-free cells were prepared from sternal cartilages of 17-day-old chick embryos, and procollagen synthesized and secreted by the cells was isolated by ion exchange chromatography on carboxymethyl cellulose and by gel filtration. The isolated protein was homogeneous by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and it appeared to consist of identical pro-α chains linked by interchain disulfide bonds. Amino acid analysis and cyanogen bromide peptide mapping of the purified procollagen demonstrated that it had structures similar to Type II collagen. The amino acid composition was also consistent with the conclusion that the peptide extensions on the pro-α chains of procollagen contained amino acid sequences not found in the collagen portion of the molecule. Segment-long-spacing aggregates were prepared from the procollagen, and aggregates demonstrated the same banding pattern as is found in segment-long-spacing aggregates prepared from Type II collagen. The segment-long-spacing aggregates from procollagen revealed, however, the presence of NH₂-terminal extensions of about 150 Å in length. In addition, the procollagen molecules contained irregularly shaped, large extension peptides at the COOH-terminal end of the molecule.     

Dynamics of β-Catenin Interactions with APC Protein Regulate Epithelial Tubulogenesis

Epithelial tubulogenesis involves complex cell rearrangements that require control of both cell adhesion and migration, but the molecular mechanisms regulating these processes during tubule development are not well understood. Interactions of the cytoplasmic protein, β-catenin, with several molecular partners have been shown to be important for cell signaling and cell–cell adhesion. To examine if β-catenin has a role in tubulogenesis, we tested the effect of expressing NH₂-terminal deleted β-catenins in an MDCK epithelial cell model for tubulogenesis. After one day of treatment, hepatocyte growth factor/scatter factor (HGF/ SF)-stimulated MDCK cysts initiated tubulogenesis by forming many long cell extensions. Expression of NH₂-terminal deleted β-catenins inhibited formation of these cell extensions. Both ΔN90 β-catenin, which binds to α-catenin, and ΔN131 β-catenin, which does not bind to α-catenin, inhibited formation of cell extensions and tubule development, indicating that a function of β-catenin distinct from its role in cadherin-mediated cell–cell adhesion is important for tubulogenesis. In cell extensions from parental cysts, adenomatous polyposis coli (APC) protein was localized in linear arrays and in punctate clusters at the tips of extensions. Inhibition of cell extension formation correlated with the colocalization and accumulation of NH₂-terminal deleted β-catenin in APC protein clusters and the absence of linear arrays of APC protein. Continued HGF/ SF treatment of parental cell MDCK cysts resulted in cell proliferation and reorganization of cell extensions into multicellular tubules. Similar HGF/SF treatment of cysts derived from cells expressing NH₂-terminal deleted β-catenins resulted in cells that proliferated but formed cell aggregates (polyps) within the cyst rather than tubules. Our results demonstrate an unexpected role for β-catenin in cell migration and indicate that dynamic β-catenin–APC protein interactions are critical for regulating cell migration during epithelial tubulogenesis.     

Prolyl 3-hydroxylase: partial characterization of the enzyme from rat kidney cortex.

The formation of 3-hydroxyproline was studied with crude rat kidney cortex extract as a source of enzyme and chick embryo tendon protocollagen and procollagen or cartilage protocollagen as a substrate. Synthesis of 3-hydroxyproline was observed with all these substrates and the formation of 3-hydroxyproline ranged up to seven residues per pro-alpha-chain. The highest rate of 3-hydroxylation took place at 20 degrees C and the reaction required Fe2+, O2,2-oxoglutarate and ascorbate. The formation of 3-hydroxyproline was affected by chain length and the conformation of the substrate, in that longer polypeptide chains proved better substrates, while the native triple-helical conformation of protocollagen or procollagen completely prevented the reaction. Formation of 3-hydroxyproline with tendon procollagen as a substrate was not inhibited by antiserum to prolyl 4-hydroxylase or by poly(L-proline) when these substances were used in concentrations which clearly inhibited 4-hydroxyproline formation with tendon protocollagen as a substrate. Furthermore, pure prolyl 4-hydroxylase did not synthesize any 3-hydroxyproline under conditions in which the crude rat kidney cortex enzyme would readily do so. The data thus strongly suggest that prolyl 3-hydroxylase and prolyl 4-hydroxylase are separate enzymes.     

Interchain disulfide bonds at the COOH-terminal end of procollagen synthesized by matrix-free cells from chick embryonic tendon and cartilage.

Matrix-free cells from tendons and cartilage of chick embryos were incubated in suspension, and the procollagens secreted by the cells were isolated in the presence of protease inhibitors. Tendon procollagen was shown to contain both NH_2? and COOH-terminal extensions and interchain disulfide bonds were located in the COOH-terminal region. A disulfide-linked fragment previously isolated after digestion of the molecule with bacterial collagenase was shown to originate from the COOH-terminal end. Cartilage procollagen was also shown to contain interchain disulfide bonds in the COOH-terminal region.     

Removal of amino-terminal and carboxy-terminal extension peptides from procollagen during synthesis of chick embryo tendon collagen

Matrix-free chick embryo tendon cells were incubated with [¹⁴C]proline for 60 minutes and protein synthesis was stopped by the addition of cycloheximide. Newly synthesized collagen precursors recovered in the incubation medium were mostly intact procollagen molecules which contain both amino-terminal and carboxy-terminal extensions. If the cells were further incubated for 2 hours in the presence of cycloheximide, most of the procollagen was converted to precursor molecules which were devoid of amino-terminal extensions. Removal of the carboxy-terminal extensions from procollagen was not observed. Similar experiments with intact tendons demonstrated that procollagen synthesized by the intact tissues $\text{in}\text{vitro}$ was readily converted to an intermediate form devoid of amino-terminal extensions and then to collagen. The results suggest that the removal of the amino-terminal and carboxy-terminal extensions from procollagen is catalyzed by two separate enzymic activities.     

The immunology of procollagen. I. Development of antibodies to determinants unique to the proalpha l chain

Rabbits immunized with proα1 chains derived from embryonic chick cranial bone procollagen developed antibodies directed specifically to the NH₂-terminal non-triple-helical sequence unique to the precursor chain. Antibodies were detected by a sensitive radioimmunoassay which employed radiolabeled chick proα1 chains, rabbit antisera absorbed with an excess of α1 chain, and a sheep anti-rabbit γG globulin serum. The specificity of such rabbit antisera was demonstrated by inhibition assays with unlabeled proα1 chains and with CNBr and collagenase fragments obtained from the additional sequence in the precursor chain. The availability of antibodies to this relatively immunogenic region of procollagen provides a means of assaying for the collagen precursor in biosynthetic experiments and may permit the intracellular localization of the macromolecule by antibody-labeling techniques.     

Enzymatic Probe for Accessibility of NH<Subscript>2</Subscript>-Terminal Residues in Immunoglobulins

ARGINYL-tRNA-protein transferase is a soluble enzyme from mammalian tissues which catalyses the transfer of arginine from arginyl-tRNA into peptide linkage specifically with NH₂-terminal aspartic or glutamic acid residues of protein acceptors^1,2. Molar equivalents of arginine are transferred to appropriate NH₂-terminal residues². The reaction differs in many respects from the transfer of amino-acids associated with protein synthesis de novo and is thought to be a mechanism for regulating the activity of acceptor proteins³. Many immunoglobulins possess aspartic or glutamic acid in the NH₂-terminal position and it was interesting to examine whether arginylation of such residues might result in an alteration of activity of these proteins, for there is considerable evidence that the antibody-combining site is contained in the NH₂-terminal region of both light^4–7 and heavy^8,9 chains.     

Expression, Purification, and Functional Characterization of the Serine Protease Inhibitor Neuroserpin Expressed in Drosophila S2 Cells

Neuroserpin (NS) is a serine protease inhibitor (or serpin) that is widely expressed in the developing and adult nervous systems. It has been implicated in the regulation of proteases involved in processes such as synaptic plasticity, neuronal migration, and axogenesis. To aid in the characterization of this new serpin we have established a high-level expression system in Drosophila S2 cells and developed a purification strategy to obtain neuroserpin for functional studies. Suspension cultures of S2-NS cells secreted recombinant neuroserpin into the medium. High-level expression was maintained when the cells were switched to a nonselection serum-free medium for 3-4 days to facilitate protein purification. Recombinant neuroserpin was purified by sequential chromatography on Macroprep ceramic hydroxyapatite, Type I, POROS HQ20, Resource Q, and Superdex 75 HR 10/30 media. Two secreted forms of neuroserpin were observed with molecular weights of 49 and 50 kDa which may represent alternative glycosylation at three putative N-linked glycosylation sites. Amino acid sequence analysis indicated three NH₂-terminal sequences. The major sequence was generated by cleavage at the Gly₁₈-Ala₁₉ bond consistent with removal of an 18-amino-acid signal peptide. Two further sequences were identified each with one fewer amino acids at the NH₂-terminus. All three NH₂-terminal sequences were also identified by mass spectrometric analysis of neuroserpin following trypsin digestion. Mass spectrometry also confirmed the protein had an intact carboxyl terminus while complex formation assays indicated the inhibitor was functionally active. In summary, Drosophila S2 cells offered a nonlytic stable expression system for the continual production of neuroserpin in high-density suspension cultures.     

Roles of the NH2-terminal Domains of Cardiac Ryanodine Receptor in Ca2+ Release Activation and Termination

The NH₂-terminal region (residues 1–543) of the cardiac ryanodine receptor (RyR2) harbors a large number of mutations associated with cardiac arrhythmias and cardiomyopathies. Functional studies have revealed that the NH₂-terminal region is involved in the activation and termination of Ca²⁺ release. The three-dimensional structure of the NH₂-terminal region has recently been solved. It is composed of three domains (A, B, and C). However, the roles of these individual domains in Ca²⁺ release activation and termination are largely unknown. To understand the functional significance of each of these NH₂-terminal domains, we systematically deleted these domains and assessed their impact on caffeine- or Ca²⁺-induced Ca²⁺ release and store overload-induced Ca²⁺ release (SOICR) in HEK293 cells. We found that all deletion mutants were capable of forming caffeine- and ryanodine-sensitive functional channels, indicating that the NH₂-terminal region is not essential for channel gating. Ca²⁺ release measurements revealed that deleting domain A markedly reduced the threshold for SOICR termination but had no effect on caffeine or Ca²⁺ activation or the threshold for SOICR activation, whereas deleting domain B substantially enhanced caffeine and Ca²⁺ activation and lowered the threshold for SOICR activation and termination. Conversely, deleting domain C suppressed caffeine activation, abolished Ca²⁺ activation and SOICR, and diminished protein expression. These results suggest that domain A is involved in channel termination, domain B is involved in channel suppression, and domain C is critical for channel activation and expression. Our data shed new insights into the structure-function relationship of the NH₂-terminal domains of RyR2 and the action of NH₂-terminal disease mutations.     

The Diphtheria Toxin Channel-forming T Domain Translocates Its Own NH2-Terminal Region Across Planar Bilayers

The T domain of diphtheria toxin, which extends from residue 202 to 378, causes the translocation of the catalytic A fragment (residues 1–201) across endosomal membranes and also forms ion-conducting channels in planar phospholipid bilayers. The carboxy terminal 57-amino acid segment (322–378) in the T domain is all that is required to form these channels, but its ability to do so is greatly augmented by the portion of the T domain upstream from this. In this work, we show that in association with channel formation by the T domain, its NH₂ terminus, as well as some or all of the adjacent hydrophilic 63 amino acid segment, cross the lipid bilayer. The phenomenon that enabled us to demonstrate that the NH₂-terminal region of the T domain was translocated across the membrane was the rapid closure of channels at cis negative voltages when the T domain contained a histidine tag at its NH₂ terminus. The inhibition of this effect by trans nickel, and by trans streptavidin when the histidine tag sequence was biotinylated, clearly established that the histidine tag was present on the trans side of the membrane. Furthermore, the inhibition of rapid channel closure by trans trypsin, combined with mutagenesis to localize the trypsin site, indicated that some portion of the 63 amino acid NH₂-terminal segment of the T domain was also translocated to the trans side of the membrane. If the NH₂ terminus was forced to remain on the cis side, by streptavidin binding to the biotinylated histidine tag sequence, channel formation was severely disrupted. Thus, normal channel formation by the T domain requires that its NH₂ terminus be translocated across the membrane from the cis to the trans side, even though the NH₂ terminus is >100 residues removed from the channel-forming part of the molecule.     

A Simple Colorimetrie Assay for Aspartate Aminotransferase

γ-Glutamylmethylamide (γ-GMA) synthetase was detected in crude extracts of Methylophaga sp. AA-30, but neither methylamine dehydrogenase nor N-methylglutamate dehydrogenase was observed. A large amount of γ-GMA was accumulated in the cells when the growth on methanol-methylamine was inhibited with iodoacetate, but the accumulation was not observed in the cells grown on methanol-(NH₄)₂SO₄. It is thought that γ-GMA is a metabolic intermediate of the methylamine-dissimilating pathway in the bacterium. In addition, γ-GMA-dissimilating enzymes were found in methylamine-grown cells. The enzymes, which consisted of H protein and L protein, required α-ketoglutaric acid, Mg²⁺ or Mn²⁺, and ammonia as a cofactor. Although the enzyme catalyzed the formation of glutamate from γ-GMA, it did not catalyze the formation of N-methylglutamate. Consequently, in this bacterium, methylamine seems to be metabolized through a different pathway from the N-methylglutamate pathway.     

70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) localizes to endoplasmic reticulum not peroxisomes, and NH2-terminal hydrophobic property determines the subcellular localization of ABC subfamily D proteins

70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) is a member of ATP-binding cassette (ABC) protein subfamily D. ABC subfamily D proteins are also known as peroxisomal ABC proteins. Therefore, P70R is thought to be a peroxisomal membrane protein. However, the subcellular localization of P70R is not extensively investigated. In this study, we transiently expressed P70R in fusion with HA (P70R-HA) in CHO cells and examined subcellular localization by immunofluorescence. Surprisingly, P70R-HA was localized to the endoplasmic reticulum (ER), not to peroxisomes. To examine the ER-targeting property of P70R, we expressed various NH₂-terminal deletion constructs of P70R. Among the NH₂-terminal deletion constructs, mutant proteins starting with hydrophobic transmembrane segment (TMS) were localized to ER, but the ones containing the NH₂-terminal hydrophilic cytosolic domain were not. ABC subfamily D proteins destined for peroxisomes have NH₂-terminal hydrophilic region adjacent to TMS1. However, only P70R lacks the region and is translated with NH₂-terminal hydrophobic TMS1. Furthermore, attachment of the NH₂-terminal hydrophilic domain to the NH₂-terminus of P70R excluded P70R from the ER-targeting pathway. These data suggest that P70R resides in the ER but not the peroxisomal membranes, and the hydrophobic property of NH₂-terminal region determines the subcellular localization of ABC subfamily D proteins.     

Localization and partial composition of the oligosaccharide units on the propeptide extensions of type I procollagen

Type I procollagen secreted by matrix-free chick embryo tendon cells was labeled with L-[3,3'-3H] cystine and purified by DEAE-cellulose chromatography. After bacterial collagenase digestion, the NH2- and COOH-terminal propeptides were partially characterized by ion exchange chromatography and gel filtration. Similar experiments were then conducted after labeling with either D-[6-3H] glucosamine, D-[2-3H] mannose, or D-[U-14C] glucose. On the basis of these studies and subsequent carbohydrate analysis, it was concluded that the COOH-terminal peptide contained greater than 90% of the radioactive carbohydrate which consisted predominantly of glucosamine and mannose with traces of galactosamine and galactose. Only radioactive glucosamine could be detected in the NH2-terminal propeptide. Under conditions which inhibit hydroxylation of lysine and glycosylation of hydroxylysine, unhydroxylated procollagen (protocollagen) could still be labeled with [3H] glucosamine and [3H] mannose. This suggested that glycosylation of the propeptides is at least initiated at the level of the rough endoplasmic reticulum.     

Archenteron formation induced by ascorbate and alpha-ketoglutarate in sea urchin embryos kept in SO2- 4 -free artificial seawater

In permanent blastulae of the sea urchin, which were obtained by culture in SO^2?₄-free artificial seawater from the time of fertilization, ascorbate and α-ketoglutarate, activators of protocollagen proline hydroxylase, induced the formation of archenteron. By adding either ascorbate or α-ketoglutarate to the SO^2?₄-free culture at 12 hr of fertilization, spherical embryos with archenteron were obtained by successive 12 hr cultures at 20°C. The embryos thus obtained did not develop to plutei. Archenteron formation induced by these compounds in SO^2?₄-free-cultured embryos, as well as in the normal embryos, was inhibited by α,α′-dipyridyl, an inhibitor of protocollagen proline hydroxylase. Glutamate, malate, citrate, and fumarate did not stimulate archenteron formation in SO^2?₄-free cultured embryos. In the SO^2?₄-free-cultured embryos exposed to [¹⁴C]proline, considerable radioactivity was found in hot trichloroacetic acid-extractable proteins but the radioactivity of [¹⁴C]hydroxyproline residue, produced by hydroxylation of proline residue of protocollagen, was markedly lower than that in normal embryos. In the presence of ascorbate and α-ketoglutarate, the radioactivity of [¹⁴C]hydroxyproline residue became high and was lowered by α,α′-dipyridyl. Archenteron formation induced by ascorbate and α-ketoglutarate in the embryos kept in SO^2?₄-free artificial seawater probably results from the stimulated protocollagen hydroxylation.     

Initiation of ovalbumin synthesis in chicken oviduct magnum: Transient labeling of the NH2-terminus by methionine

The incorporation of [³⁵S]methionine into ovalbumin, a protein containing NH₂-terminal N-acetylglycine, has been studied in chicken oviduct magnum cells. The purification of [³⁵S]methionine-labeled ovalbumin from total oviduct proteins was accomplished by dialysis of a crude extract at pH 3.6 followed by chromatography on carboxymethyl cellulose. The radioactive ovalbumin eluted from the column in three peaks (P₀, P₁, and P₂-containing 0, 1, and 2 moles of phosphate, respectively, per mole of ovalbumin). The kinetics of labeling of peaks P₀ and P₁ showed that the ratio of radioactivity in NH₂-terminal methionine to total incorporation was greater at 2 min of labeling than at later times. The transient labeling of the NH₂-terminus of ovalbumin with methionine indicates that methionine is the initiator amino acid for the synthesis of this protein, which in its mature form contains NH₂-terminal N-acetylglycine.     

Pleckstrin Associates with Plasma Membranes and Induces the Formation of Membrane Projections: Requirements for Phosphorylation and the NH2-terminal PH Domain

Pleckstrin homology (PH) domains are sequences of ~100 amino acids that form “modules” that have been proposed to facilitate protein/protein or protein/lipid interactions. Pleckstrin, first described as a substrate for protein kinase C in platelets and leukocytes, is composed of two PH domains, one at each end of the molecule, flanking an intervening sequence of 147 residues. Evidence is accumulating to support the hypothesis that PH domains are structural motifs that target molecules to membranes, perhaps through interactions with Gβγ or phosphatidylinositol 4,5-bisphosphate (PIP₂), two putative PH domain ligands. In the present studies, we show that pleckstrin associates with membranes in human platelets. We further demonstrate that, in transfected Cos-1 cells, pleckstrin associates with peripheral membrane ruffles and dorsal membrane projections. This association depends on phosphorylation of pleckstrin and requires the presence of its NH₂-terminal, but not its COOH-terminal, PH domain. Moreover, PH domains from other molecules cannot effectively substitute for pleckstrin's NH₂terminal PH domain in directing membrane localization. Lastly, we show that wild-type pleckstrin actually promotes the formation of membrane projections from the dorsal surface of transfected cells, and that this morphologic change is similarly PH domain dependent. Since we have shown previously that pleckstrin-mediated inhibition of PIP₂ metabolism by phospholipase C or phosphatidylinositol 3-kinase also requires pleckstrin phosphorylation and an intact NH₂-terminal PH domain, these results suggest that: (a) pleckstrin's NH₂terminal PH domain may regulate pleckstrin's activity by targeting it to specific areas within the cell membrane; and (b) pleckstrin may affect membrane structure, perhaps via interactions with PIP₂ and/or other membrane-bound ligands.     

Identification of interleukin-8 converting enzyme as cathepsin L

IL-8 is produced by various cells, and the NH₂-terminal amino acid sequence of IL-8 displays heterogeneity among cell types. The mature form of IL-8 has 72 amino acids (72IL-8), while a precursor form (77IL-8) of IL-8 has five additional amino acids to the 72IL-8 NH₂-terminal. However, it has been unclear how IL-8 is processed to yield the mature form. In this study, converting enzyme was purified as a single 31-kDa band on silver-stained polyacrylamide gel from 160 l of cultured fibroblast supernatant by sequential chromatography. NH₂-terminal amino acid sequence analysis revealed a sequence, EAPRSVDWRE, which was identified as a partial sequence of cathepsin L. Polyclonal antibodies raised against cathepsin L recognized the purified converting enzyme on Western blot. Moreover, human hepatic cathepsin L cleaved 77IL-8 between Arg⁵ and Ser⁶, which is the same cleavage site as the putative converting enzyme, resulting in 72IL-8 formation. These data indicate that the converting enzyme of the partially purified fraction of the human fibroblast culture supernatant was cathepsin L. Furthermore, 72IL-8 was sevenfold more potent than 77IL-8 in a neutrophil chemotaxis assay. These results show that cathepsin L is secreted from human fibroblasts in response to external stimuli and plays an important role in IL-8 processing in inflammatory sites.     

Effect of the arginine analog,canavanine, on the synthesis and secretion of procollagen

Canavanine was shown to competitively inhibit the activation of arginine when tested with tRNA and synthetases prepared from whole chick embryos. The canavanine has no effect when tested with other amino acids. The K_m for arginine was 2.5 μm and the K_i for canavanine was 35 μm. When fibroblasts from embryonic chick tendons were incubated with [³H]arginine and increasing concentrations of canavanine, there was a progressive decrease in the incorporation of [³H]arginine so that at 3 mm the incorporation into nondialyzable protein was only 14% of the control. A much smaller decrease in the incorporation of other radioactive amino acids was observed. Amino acid analysis of proteins isolated from cells incubated with canavanine showed conclusively that the analog was incorporated. When the cells were incubated with [¹⁴C]proline or [³H]glycine and 3 mm canavanine, the labeled procollagen containing the canavanine was secreted more slowly than normal and accumulated intracellularly. The retained procollagen chains were normally hydroxylated, disulfide linked, and triple helical. However, slab gel electrophoresis in sodium dodecyl sulfate demonstrated that they migrated with a lower mobility than control procollagen chains. We postulate that incorporation of canavanine inhibits normal proteolytic processing of signal sequences resulting in delayed secretion of the procollagen.