Life behind cell walls: paradigm lost, paradigm regained.

This review of the living cell wall and its protein components is in two parts. The first is anecdotal. A personal account spanning over 40 years research may perhaps be an antidote to one stereotypical view of scientists as detached and humorless. The second part deals with the meaning of function, particularly as it applies to hydroxyproline-rich glycoproteins. Function is a difficult word to define objectively. However, with help from such luminaries as Humpty Dumpty: "A word means what I want it to mean, neither more nor less," and Wittgenstein: "Giving examples of usage ... is the only way to talk about meaning," it is possible to construct a ziggurat representing increasingly complex levels of organization from molecular structure to ecology. Forty years ago I suggested that hydroxyproline-rich structural proteins played a key role in cell wall functioning. But because the bulk of the wall is carbohydrate, there has been an understandable resistance to paradigm change. Expansins, paradoxically, contribute greatly to this resistance because their modus operandi as cell-wall-loosening proteins is based on the idea that they break hydrogen bonds between polysaccharide chains allowing slippage. However, this view is not consistent with the recent discovery [Grobe et al. (1999) Eur. J. Biochem 263: 33-40] that beta-expansins may be proteases, as it implies that the extensin network is not a straightjacket but a substrate for expansin in muro. Such a direct role for extensins in both negative and positive regulation of cell expansion and elongation may constitute a major morphogenetic mechanism operating at all levels of plant growth and development.

Isolation, characterization and immunolocalization of a novel, modular tomato arabinogalactan-protein corresponding to the LeAGP-1 gene.

Arabinogalactan-proteins (AGPs) are a family of hydroxy-proline-rich glycoproteins implicated to function in plant growth and development. This report focuses on a novel, modular AGP found in tomato, LeAGP-1, which was predicted by DNA cloning and herein verified at the protein level as a major AGP component. LeAGP-1 was isolated from tomato suspension-cultured cells and verified to be an AGP by precipitation with (beta-D-galactosyl)3 Yariv phenylglycoside and by amino acid composition analysis. Furthermore, LeAGP-1 was determined to correspond to LeAGP-1 clones based on three criteria: (1) amino acid composition identity, (2) amino acid sequence identity, and (3) specific immunoreactivity of glycosylated and deglycosylated LeAGP-1 with an antibody developed against the highly basic subdomain predicted from LeAGP-1 clones. The antibody was also used to immunolocalize LeAGP-1 in tomato to the cell surface of suspension-cultured cells, maturing metaxylem elements in young internodes and petioles, and stylar transmitting tissue cells. At the subcellular level, LeAGP-1 immunolocalized to the cell walls of these particular cells as well as to intercellular spaces between stylar transmitting tissue cells. LeAGP-1 now emerges as one of the most comprehensively studied AGPs in terms of (1) characterization at the genomic DNA, cDNA and protein levels, (2) known organ-specific and developmentally regulated mRNA expression patterns, (3) development of an antibody against a unique, peptide subdomain which specifically recognizes LeAGP-1 in its glycosylated and deglycosylated states, and (4) immunolocalization of a single, well-defined AGP molecule at the tissue and subcellular levels.

Isolation of pl 4.6 extensin peroxidase from tomato cell suspension cultures and identification of Val-Tyr-Lys as putative intermolecular cross-link site.

Extensins and kindred hydroxyproline-rich glycoproteins occur in dicot cell walls mainly as insoluble integral components that may form an intermolecularly cross-linked network interpenetrated by other polymers. Extensins also occur in muro as a small pool of soluble monomeric precursors to network extensin. These precursors were prepared in milligram quantities by salt elution from the surface of intact cells grown as tomato suspension cultures. Based on an FPLC (Superose-6) gel filtration assay of cross-linked extensin oligomers, a pl 4.6 extensin cross-linking peroxidase isozyme was partially purified from the culture growth medium. Purification involved: volume reduction, ultracentrifugation to remove pectin and co-adsorbed cationic peroxidase, followed by chromatography of anionic extensin peroxidase (pl 4.6) on DEAE-Trisacryl and TSK-gel DEAE-5PW columns. With tomato P1 extensin as substrate and 60 microM H2O2 as co-substrate, at 23 degrees pl 4.6 extensin peroxidase gave a Km of 0.22 mM P1 and a Vmax 0f 70 mumol P1 cross-linked min-1mg-1 enzyme, at the optimum pH 5.5. Assayed with 12 different extensins from representative monocots, dicots, and gymnosperms, the pl 4.6 isozyme cross-linked highly selectively, indicating two natural groups: cross-linking or CL-extensins and non-cross-linking or NCL-extensins. CL-extensins contained the X-Hyp-Val-Tyr-Lys motif and were also highly glycosylated. However, the simplest motif common to CL-extensins but absent from NCL-extensins was Val-Tyr-Lys. Thus, peroxidative coupling of extensin monomers and resistance of the resultant oligomers to depolymerization by anhydrous HF suggests that the intermolecular cross-link involves tyrosine or lysine.

Extensin: repetitive motifs, functional sites, post-translational codes, and phylogeny.

Homologous hydroxyproline-rich glycoproteins (HRGPs) of the plant extracellular matrix include extensins, repetitive proline-rich proteins (RPRPs), some nodulins, gum arabic glycoprotein (GAGP), arabinogalactan-proteins (AGPs), and chimeric proteins such as potato lectin which contain an extensin module fused to a lectin. The key to the role of HRGPs in cell wall self-assembly and cell extension lies in their chemistry, which is dependent on extensive post-translational modifications (PTMs): hydroxylation, glycosylation, and cross-linking. Repetitive peptide motifs characterize HRGPs. One or more repetitive peptide motifs and their variants, singly or in combination, may constitute functional sites involved in various aspects of cell wall assembly, as follows: (i) X-Hypn including Ser-Hyp4 (arabinosylation site, molecular rigidity, and reptation). (ii) Pro-Hyp-Val-Tyr-Lys and variants (putative intermolecular cross-links, adhesion, cohesion, and possible beta-turns). (iii) Tyr-X-Tyr-Lys (intramolecular isodityrosine [IDT] cross-links increase molecular rigidity and hydrophobicity). (iv) (Glyco)peptide palindromes (centrosymmetric domains: putative self-assembly nucleation sites). (v) Ionic interaction sites (protein-protein and protein-carbohydrate cross-links). (vi) Hyp and Ser glycosylation sites (enhance conformational stability and molecular recognition). (vii) Extensin modules in chimeric proteins (e.g. solanaceous lectins). Rules for the post-translational modifications are emerging: (i) Hydroxylation of proline residues may depend on multiple, sequence-specific prolyl hydroxylases rather than on a single (polyproline-II) conformation-dependent enzyme. Furthermore, Lys-Pro, Tyr-Pro, and Phe-Pro are not hydroxylated, while Pro-Val is always. (ii) Contiguity of Hyp residues probably determines the extent of Hyp glycosylation, blocks of tetrahydroxyproline (Hyp4) being the most highly arabinosylated, while single non-contiguous Hyp residues are rarely arabinosylated, although they are likely attachment sites for the larger arabinogalactan substituents of gum arabic glycoprotein and arabinogalactan-proteins. (iii) While intramolecular cross-links involve IDT, unidentified intermolecular cross-links most likely involve the Val-Tyr-Lys motif (perhaps also Val-Lys-Pro-Tyr-His-Pro), probably as an adduct between Tyr and Lys catalyzed in vitro by a pI 4.6 extensin cross-linking peroxidase. Thus, we can classify HRGPs functionally as either cross-linking or non-cross-linking, i.e. CL- or NCL-extensins. Their protistan origin obscures the phylogenetic affinities of a single extensin-HRGP family due to their sequence divergence. We propose a phylogenetic series ranging from the minimally glycosylated basic RPRPs to the highly glycosylated acidic AGPs. Furthermore, based on similarities between dicots and gymnosperm extensins, and their marked difference from graminaceous monocot extensins, graminaceous monocot and dicot lines may have diverged as early as the progymnosperms.(ABSTRACT TRUNCATED AT 400 WORDS)

A Histidine-Rich Extensin from Zea mays Is an Arabinogalactan Protein.

Earlier we isolated a threonine-rich extensin from maize (Zea mays). Here, we report that maize cell suspension cultures yield a new extensin rich in histidine (HHRGP) that also has characteristics of arabinogalactan proteins (AGPs). Thus, chymotryptic peptide maps of anhydrous hydrogen fluoride (HF)-deglycosylated HHRGP showed repetitive motifs related to both extensins and AGPs as follows. HHRGP contains Ala-Hyp(3) and Ala-Hyp(4) repeats that may be related to the classical dicot Ser-Hyp(4) extensin motif by the single T --> G (Ser --> Ala) base change. Furthermore, HHRGP also contains the repetitive motif Ala-Hyp-Hyp-Hyp-His-Phe-Pro-Ser-Hyp-Hyp related to the Ser-Hyp(4)-Ser-Hyp-Ser-Hyp(4) motif of P3-type dicot extensin. However, HHRGP also has AGP characteristics, notably an elevated alanine content, near sequence identity with the known Lolium AGP peptide Ser-Hyp-Hyp-Ala-Pro-Ala-Pro, the putative presence of glucuronoarabinogalactan, and precipitation by Yariv antigen, but beta-elimination of arabinogalactan indicates its O-linkage to serine rather than the characteristic O-hydroxyproline link of other AGPs. Although HHRGP might be a "chimera" of two different proteins, i.e. an extensin and an AGP, this is unlikely because one can account for the apparent chimera by the codon relationships of the five common hydroxyproline-rich glycoprotein amino acid residues, Ser, Pro, Thr, Ala (TCx, CCx, ACx, GCx) and histidine (CAT or CAC), which facilitate interconversion of major motifs by single point mutations. Thus, we propose that the extensin family of wall proteins consists of a highly diversified phylogenetic series ranging from basic minimally glycosylated repetitive pro-rich proteins to the highly glycosylated acidic AGPs. To relate this diversity of form and function at the molecular level, we identified putative functional domains hypothetically involved in properties such as reptation, recognition, adhesion, intermolecular cross-linkage, and self-assembly. Not previously noted, peptide palindromes feature prominently in HHRGP: Hyp-Hyp-Ala-Ala-Asn-Ala-Ala-Hyp-Hyp and Hyp-Hyp-Hyp-His-His-His-Hyp-Hyp-Hyp; in P3: Hyp(4)-Ser-Hyp-Ser-Hyp(4), and in other extensins. Such palindromes would enhance glycoprotein stereoregularity, thereby possibly promoting quasicrystalline interactions between wall components.

A gymnosperm extensin contains the serine-tetrahydroxyproline motif.

The extensin family is a diverse group of hydroxyproline-rich glycoproteins located in the cell wall and characterized by repetitive peptide motifs glycosylated to various degrees. The origin of this diversity and its relationship to function led us earlier to compare extensins of the two major groups of angiosperms from which we concluded that the highly glycosylated Ser-Hyp(4) motif was characteristic of advanced herbaceous dicots, occurring rarely or not at all in a representative graminaceous monocot (Zea mays) and a chenopod (Beta vulgaris) representative of primitive dicots. Because these results could arise either from loss or acquisition of a characteristic feature, we chose a typical gymnosperm representing seed-bearing plants more primitive than the angiosperms. Thus, salt eluates of Douglas fir (Pseudotsuga menziesii) cell suspension cultures yielded two monomeric extensins differing in size and composition. The larger extensin reported earlier lacked the Ser-Hyp(4) motif, was rich in proline and hydroxyproline, and contained peptide motifs similar to the dicot repetitive proline-rich proteins. The smaller extensin monomer reported here (Superose-6 peak 2 [SP2]) was compositionally similar to typical dicot extensins such as tomato P1, mainly consisting of Hyp, Thr, Ser, Pro, Val, Tyr, Lys, His, abundant arabinose, and a small but significant galactose content. A chymotryptic peptide map (on Hamilton PRP-1) of anhydrous hydrogen fluoride-deglycosylated SP2 yielded eight peptides sequenced after further purification on a high-resolution fast-sizing column (polyhydroxyethyl aspartamide; Poly LC). Significantly, two of the eight peptides contained the Ser-Hyp(4) motif, consistent both with the SP2 amino acid composition as well as the presence of hydroxyproline tetraarabinoside as a small (4% of total Hyp) component of the hydroxyproline arabinoside profile; thus, hydroxyproline tetraarabinoside corroborates the presence of Ser-Hyp(4), in agreement with our earlier observation that Hyp contiguity and Hyp glycosylation are positively correlated. Interestingly, other peptide sequences indicate that SP2 contains motifs such as Ser-Hyp(3)-Thr-Hyp-Tyr, Ser-Hyp(4)-Lys, and (Ala-Hyp)(n) repeats that are related to and typify dicot extensins P1, P3, and arabinogalactan proteins, respectively. Overall, these peptide sequences confirm our previous prediction that Ser-Hyp(4) is indeed an ancient motif and also strongly support our suggestion that the extensins comprise an extraordinarily diverse, but nevertheless phylogenetically related, family of cell wall hydroxyproline-rich glycoproteins.

A repetitive proline-rich protein from the gymnosperm douglas fir is a hydroxyproline-rich glycoprotein.

Intact cell elution of suspension cultures derived from Douglas fir, Pseudotsuga menziesii (Mirbel) Franco, yielded two extensin monomers, the first hydroxyproline-rich glycoproteins (HRGPs) to be isolated from a gymnosperm. These HRGPs resolved on Superose-6 gel filtration. The smaller monomer was compositionally similar to angiosperm extensins like tomato P1. The larger monomer had a simple composition reminiscent of repetitive proline-rich proteins (RPRPs) from soybean cell walls and contained proline, hydroxyproline, and sugar; hence designated a proline-hydroxyproline-rich glycoprotein (PHRGP). The simple composition of the PHRGP implied a periodic structure which was confirmed by the simple chymotryptic map and 45-residue partial sequence of the major proline-hydroxyproline-rich glycoprotein chymotryptide 5: Lys-Pro-Hyp-Val-Hyp-Val-Ile-Pro-Pro-Hyp-Val-Val-Lys-Pro-Hyp-Hyp-Val- Tyr-Lys-Pro-Hyp-Val-Hyp-Val-Ile-Pro-Pro-Hyp-Val-Val-Lys-Pro-Hyp-Hyp- Val-Tyr-Lys-Ile-Pro-Pro(Hyp)-Val-Ile-Lys-Pro. Proline-hydroxyproline-rich glycoprotein chymotryptide 5 contained an 18-residue tandem repeat devoid of tetra(hydroxy)-proline or serine; it also contained two instances of the five-residue motif Hyp-Hyp-Val-Tyr-Lys and five of the general Pro-Pro-X-X-Lys motif, thereby establishing its homology with typical angiosperm RPRPs and extensins from tomato, petunia, carrot, tobacco, sugar beet, and Phaseolus. Unlike the nonglycosylated soybean RPRP, the highly purified Douglas fir PHRGP was lightly glycosylated, confirmed by a quantitative hydroxyproline glycoside profile, indicating that extensins can range from highly glycosylated hydroxyproline to little or no glycosylated hydroxyproline. Comparison of extensin sequence data strongly indicates that a major determinant of hydroxyproline glycosylation specificity is hydroxyproline contiguity: extensins with tetrahydroxyproline blocks are very highly arabinosylated (>90% hydroxyproline glycosylated), tri- and dihydroxyproline are less so, and single hydroxyproline residues perhaps not at all. Despite high yields of extensins eluted from intact cells, the Douglas fir cell wall itself was hydroxyproline poor yet remarkably rich in protein (>20%), again emphasizing the existence of other structural cell wall proteins that are neither HRGPs nor glycine-rich proteins.

Epitope expression on the breast epithelial mucin.

Multiple epitope expression on the breast epithelial mucin was explored using a panel of monoclonal antibodies (MoAbs) created against milk and breast tissue preparations, against blood group determinants, and against other non-breast epithelial mucins. Since the breast epithelial mucin is now used in both diagnostic and therapeutic modalities for breast cancer, and also because altered or incomplete glycosylation in varying degrees is expected in breast carcinoma tissue, the antigenic target used here was the native mucin and sequential stages of deglycosylation introduced to it by HF treatment. Partial deglycosylation increased exposure of core peptide amino acid sequences increasing MoAb binding generally, while it either decreased or occasionally increased binding of blood group oligosaccharides. Cross reactivity of MoAbs to other mucins was low with the breast epithelial mucin (BEM). The study of the affinity binding constants of some of the anti-BEM peptide MoAbs predicted carbohydrate participation in their epitope structure. The identification of different epitopes on the BEM, investigations on their possible epitopic structure, and the study of MoAb binding during different stages of glycosylation of the molecule leads to knowledge on the contribution of carbohydrates to their epitopes and strengthens the ability to understand their performance in their diverse possible applications in breast cancer diagnosis, prognosis, and therapy.

Molecular cloning of rat intestinal mucin. Lack of conservation between mammalian species.

We have prepared antisera to deglycosylated rat intestinal mucin and used it to obtain immunoreactive clones from a rat jejunum cDNA library. Four of these clones were sequenced, and all were found to be partial cDNAs that contained 18-base pair tandem repeats characteristic of a mucin. These cDNAs encoded a repetitive peptide with a consensus sequence of TTTPDV. Thus, they bear little resemblance to either of the two human intestinal mucin cDNAs isolated previously (Gum, J. R., Byrd, J. C., Hicks, J. W., Toribara, N. W., Lamport, D. T. A., and Kim, Y. S. (1989) J. Biol. Chem. 264, 6480-6487 and Gum, J. R., Hicks, J. W., Swallow, D. M., Lagace, R. E., Byrd, J. C., Lamport, D. T. A., Siddiki, B., and Kim, Y. S. (1990) Biochem. Biophys. Res. Commun. 171, 407-415). One of these rat mucin clones, designated RMUC 176, was chosen for further analysis. This clone recognized a band of approximately 9 kilobases when used to probe RNA blots. A strong hybridization band was present using rat small intestine and colon RNA but was not detectable when RNA isolated from heart, liver, or kidney was tested. The RMUC 176 clone and the two previously isolated human intestinal mucin cDNA clones were used to probe blots prepared from BamHI-digested DNA of various species. Here, the human probes detected fragments present only in human and chimpanzee DNA, whereas the RMUC 176 clone recognized fragments only in rat and mouse DNA. Thus, the repetitive portions of intestinal mucin genes are apparently not well conserved between phylogenetically distant species.

Gum arabic glycoprotein is a twisted hairy rope : a new model based on o-galactosylhydroxyproline as the polysaccharide attachment site.

Separation of the wound exudate from Acacia senegal (L.) Willd., "gum arabic," on a preparative Superose-6 column gave two major fractions: a high molecular weight gum arabic glycoprotein (GAGP) containing about 90% carbohydrate and a lower molecular weight heterogenous gum arabic polysaccharide fraction. Hydrogen fluoride-deglycosylation of GAGP gave a large ( approximately 400 residue) hydroxyproline-rich polypeptide backbone (dGAGP). Alkaline hydrolysis of GAGP showed that most of the carbohydrate was attached to the polypeptide backbone as small ( approximately 30 residue) hydroxyproline (Hyp)-polysaccharide substituents. After partial acid hydrolysis of the Hyp-polysaccharide fraction we identified O-galactosylhydroxyproline as the glycopeptide linkage, identical with that of hydroxyproline-rich arabinogalactan-proteins (AGPs). However, unlike the acidic alanine-rich AGPs, GAGP is basic and notably deficient in alanine. Thus, while the GAGP polypeptide backbone more closely resembles that of the Hyp-rich cell wall protein extensin, the GAGP polysaccharide sidechains resemble AGPs. Possibly all three proteins comprise a phylogenetically related extensin superfamily of extended rod-like macromolecules. The "wattle-blossom" model for AGP and gum arabic predicts a few large polysaccharide substituents along the polypeptide backbone of a spheroidal macromolecule. On the contrary, our data imply a rodlike molecule with numerous small polysaccharide substituents (attached to 24% of the Hyp residues), regularly arranged along a highly periodic polypeptide backbone based, hypothetically, on a 10 to 12 residue repetitive peptide motif. Thus, a simple statistical model of the gum arabic glycoprotein predicts a repeating polysaccharide-peptide subunit of about 7 kilodaltons. The small polysaccharide substituents will maximize intramolecular hydrogen bonding if aligned along the long axis of the molecule, forming in effect a twisted hairy rope. Electron micrographs of rotary shadowed GAGP molecules support that prediction and may also explain how such apparently large molecules can exit the cell by endwise reptation through the small pores of the primary cell wall.

Relationship of pancreatic cancer apomucin to mammary and intestinal apomucins.

Pancreatic cancer mucins have several carbohydrate antigens that are potentially useful in the detection of pancreatic cancers, but little is known about the core polypeptides of pancreatic cancer mucins. In this study, purified mucin from SW1990 pancreatic cancer xenografts was deglycosylated by treatment with hydrogen fluoride to give pancreatic cancer apomucin. Consistent with near-complete removal of carbohydrate, the apomucin had 10- to 70-fold decreased binding of lectins and, unlike the native mucin, served as an acceptor for polypeptidyl N-acetylgalactosaminyl transferase. Antibodies prepared against the apomucin did not bind to native mucin, and antibodies that bound to native mucin did not bind to apomucin. On the basis of cross-reaction with deglycosylated colon cancer mucin and intestinal mucin repeat peptide, apomucins from SW1990 pancreatic cancer xenografts contain the intestinal mucin repeat peptide. On the basis of binding of breast cancer-reactive monoclonal antibodies 139H2, DF3, and HMFG-2, apomucins from SW1990 pancreatic cancer xenografts also have the mammary mucin repeat peptide. Using complementary DNA probes specific for intestinal mucin and breast mucin sequences, both types of apomucin mRNA were detected in nude mouse xenografts of SW1990 cells. In immunohistochemical staining, antibody against deglycosylated SW1990 mucin stained normal breast and pancreas but not normal colon. Some pancreatic and mammary cancers and most colonic cancers, however, were stained by antibodies against both intestinal apomucin and mammary apomucin. We conclude that pancreatic cancers can produce mucins with the intestinal repeat peptide as well as those with mammary repeat peptide sequences.

Adaptation and growth of tomato cells on the herbicide 2,6-dichlorobenzonitrile leads to production of unique cell walls virtually lacking a cellulose-xyloglucan network.

Suspension-cultured cells of tomato (Lycopersicon esculentum VF 36) have been adapted to growth on high concentrations of 2,6-dichlorobenzonitrile, an herbicide which inhibits cellulose biosynthesis. The mechanism of adaptation appears to rest largely on the ability of these cells to divide and expand in the virtual absence of a cellulose-xyloglucan network. Walls of adapted cells growing on 2,6-dichlorobenzonitrile also differ from nonadapted cells by having reduced levels of hydroxyproline in protein, both in bound and salt-elutable form, and in having a much higher proportion of homogalacturonan and rhamnogalacturonan-like polymers. Most of these latter polymers are apparently cross-linked in the wall via phenolic-ester and/or phenolic ether linkages, and these polymers appear to represent the major load-bearing network in these unusual cell walls. The surprising finding that plant cells can survive in the virtual absence of a major load-bearing network in their primary cell walls indicates that plants possess remarkable flexibility for tolerating changes in wall composition.

Molecular cloning of cDNAs derived from a novel human intestinal mucin gene.

A human small intestinal lambda gt11 cDNA library was screened with antibodies to deglycosylated small intestinal mucin. Four partial cDNA clones were isolated that define a novel human mucin gene. These include two partial cDNA clones, SIB 124 and SIB 139, that contain 51 nucleotide tandem repeats which encode a seventeen amino acid repetitive peptide with a consensus sequence of HSTPSFTSSITTTETTS. SIB 139 hybridized to messages produced by small intestine, colon, colonic tumors and also by high mucin variant LS174T colon cancer cells. The gene from which cDNAs SIB 124 and SIB 139 are derived (proposed name MUC 3) maps to chromosome 7, distinct from other known human mucin genes.

Structure of the Threonine-Rich Extensin from Zea mays.

Chymotryptic digestion of a threonine-rich hydroxyproline-rich glycoprotein (THRGP) purified from the cell surface of a Zea mays cell suspension culture gave a peptide map dominated by the hexadecapeptide TC5: Thr-Hyp-Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys-Pro-Thr-Hyp-Hyp-Thr-Tyr, in which the repetitive motif Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys is homologous with the dominant decamer of P1-type dicot extensins: Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, modified by a Lys for Hyp substitution at residue 3, a Val-Tyr deletion at residues 8 and 9, and incomplete post-translational modification of proline residues. One of the minor peptides (TC1) contained the 8-residue sequence: Thr-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Tyr corresponding to the C-terminal tail (judging from the recently isolated maize cDNA clone MC56) which is homologous with the major repetitive motif of the ;P3' class of dicot extensins. Direct peptide sequencing defined potential glycosylated regions on the THRGP corresponding to clone MC56 and showing that glycosylated and nonglycosylated domains alternate with high regularity. The THRGP is not in the polyproline-II conformation, judging from circular dichroic spectra, but nevertheless is an extended rod, from electron microscopic data. HF-solvolysis of cell walls from maize coleoptile, root, and root tip released deglycosylated THRGP detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots with high titer rabbit polyclonal antibodies raised against the intact THRGP. In a quantitative enzyme-linked immunosorbent assay, these antibodies cross-reacted 20% with tomato P1 extensin, and 18% with anhydrous hydrogen fluoride-deglycosylated P1. These results, together with other previously published data, show that maize THRGP is homologous with the dicot P1 extensins and, as such, is the first extensin isolated from a graminaceous monocot.

A chenopod extensin lacks repetitive tetrahydroxyproline blocks.

An extensin isolated from sugar beet (Beta vulgaris) cell suspension cultures fulfills all criteria for membership of the extensin family save one, notably, lack of the ;diagnostic' pentamer Ser-Hyp-Hyp-Hyp-Hyp. However, sequence analysis of the major tryptic peptides shows that sugar beet extensin shares a motif in common with tomato extensin P1 but differs by the position of an insertion sequence [X] or [Y] which, in sugar beet, splits the tetrahydroxyproline block: Ser-Hyp-Hyp-[X]-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, where [X] is [Val-His-Glu/Lys-Tyr-Pro], while in tomato the insertion sequence [Y] = [Val-Lys-Pro-Tyr-His-Pro] and, when it occurs, immediately follows the tetrahydroxyproline block: Ser-Hyp-Hyp-Hyp-Hyp-[Y]-Thr-Hyp-Val-Tyr-Lys. Based on these data we reinterpret three highly repetitive cDNA sequences, including nodulin N75 from soybean and wound-induced P33 of carrot, as extensins with split tetra(hydroxy)proline blocks.

Deglycosylation of mucin from LS174T colon cancer cells by hydrogen fluoride treatment.

Mucin from xenografts of LS174T human colon cancer cells was treated with anhydrous HF for 1 h at 0 degree C to give a product (HFA) with over 80% of the glucosamine and hexose removed, but retaining some galactosamine, and for 3 h at room temperature to give a product (HFB) devoid of carbohydrate. Rabbit antibodies against HFA bound to HFA much more than to HFB, and bound to native mucin to an intermediate extent. Antibodies to HFB bound to HFB more than to HFA, and did not bind to native mucin. Both HFA and native mucin bound a number of lectins, but HFB did not. By SDS/polyacrylamide-gel electrophoresis and size-exclusion h.p.l.c., native mucin and HFA are of apparent molecular mass greater than 400 kDa, whereas HFB is heterogeneous and of low molecular mass. On Western blots, antibody to HFA detected both high-molecular-mass mucin and a 90 kDa protein in homogenates of LS174T cells. Antibody to HFB detected a major 70 kDa band as well as higher-molecular-mass species. In tissue sections of normal colon and colon cancers, antibody to HFA showed both cytoplasmic and extracellular staining, whereas antibody to HFB generally stained only cytoplasmic antigens. These results indicate that anti-HFB antibody is specific for apo-mucin, whereas anti-HFA antibody is specific for GalNAc-apo-mucin.

Trypsin cleaves lysylproline in a hydroxyproline-rich glycoprotein from Zea mays.

Although trypsin is highly specific for lysyl and arginyl bonds, some peptide bonds, such as lysylproline, are generally trypsin-resistant, with rare exceptions as reported here. Trypsin cleaved a specific Lys-Pro bond in the chymotryptic peptide: Thr-Hyp-Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys-Pro-Thr-Hyp-Hyp-Thr-Tyr isolated from a Zea mays hydroxyproline-rich glycoprotein (HRGP). The daughter peptides, Thr-Hyp-Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys and Pro-Thr-Hyp-Hyp-Thr-Tyr, show cleavage of only one of the two Lys-Pro bonds in the parent peptide. From these and other data we suggest that there are two prerequisites for Lys-Pro cleavage: First, an extended helix characteristically present in proline or hydroxyproline-rich proteins; second, flexibility in two residues flanking the Lys-Pro bond.

Molecular cloning of human intestinal mucin cDNAs. Sequence analysis and evidence for genetic polymorphism.

A human small intestine lambda gt11 cDNA library was screened using antisera prepared against the deglycosylated protein backbone of human colon cancer xenograft mucin. Three cDNAs were isolated from this screening, designated SMUC 40-42. These cDNAs were all found to contain tandem repeats of 69 nucleotides which encoded a threonine- and proline-rich protein consensus sequence of PTTTPITTTTTVTPTPTPTGTQT. RNA blots probed with one of these cDNAs, SMUC 41, exhibited large, polydisperse hybridization bands at approximately 7,600 bases. Band intensities were strongest when human small intestine, colon, and colon cancer poly(A)+ RNA was used. In vitro translation of poly(A)+ RNA from human small intestine, colon, and colon cancer cells produced a 162,000-dalton peptide that was immunoprecipitated with antibodies to deglycosylated mucin. SMUC 41 was also used to probe DNA blots, which indicated the presence of restriction fragment length polymorphisms in the intestinal mucin gene. These findings may be important in assessing the abnormal mucins found associated with several human diseases.

Enzymic cross-linkage of monomeric extensin precursors in vitro.

Rapidly growing tomato (Lycopersicon esculentum) cell suspension cultures contain transiently high levels of cell surface, salt-elutable, monomeric precursors to the covalently cross-linked extensin network of the primary cell wall. Thus, we purified a highly soluble monomeric extensin substrate from rapidly growing cells, and devised a soluble in vitro cross-linking assay based on Superose-6 fast protein liquid chromatography separation, which resolved extensin monomers from the newly formed oligomers within 25 minutes. Salt elution of slowly growing (early stationary phase) cells yielded little or no extensin monomers but did give a highly active enzymic preparation that specifically cross-linked extensin monomers in the presence of hydrogen peroxide, judging from: (a) a decrease in the extensin monomer peak on fast protein liquid chromatography gel filtration, (b) appearance of oligomeric peaks, and (c) direct electron microscopical observation of the cross-linked oligomers. The cross-linking reaction had a broad pH optimum between 5.5 and 6.5. An approach to substrate saturation of the enzyme required extensin monomer concentrations of 20 to 40 milligrams per milliliter. Preincubation with catalase completely inhibited the cross-linking reaction, which was highly dependent on hydrogen peroxide and optimal at 15 to 50 micromolar. We therefore identified the cross-linking activity as extensin peroxidase.