Linked Galactose Residues Research Articles

Galectin-3 binds selectively to the terminal, non-reducing end of β(1→4)-galactans, with overall affinity increasing with chain length.

Galactans are linear polysaccharides of β(1→4)-linked galactose residues. Although they can antagonize galectin function, the nature of their binding to galectins needs to be better defined to develop them as drugs. Here, we investigated interactions between galectin-3 (Gal-3) and a series of galactans ranging in weight average molecular weight from 670 to 7550 Da. 15N-1H HSQC NMR studies with 15N-labeled Gal-3 carbohydrate recognition domain (CRD) indicate that each of these galactans interacts primarily with residues in β-strands 4, 5 and 6 on the canonical, β-galactoside sugar binding S-face. Although these galactans also bind to full length Gal-3 (CRD plus N-terminal tail) to the same extent, it appears that binding to the S-face attenuates interactions between the CRD F-face and N-terminal tail, making interpretation of site-specific binding unclear. Following assignment of galactan 13C and 1H resonances using HSQC, HMBC and TOCSY experiments, we used 13C-1H HSQC data to demonstrate that the Gal-3 CRD binds to the terminal, non-reducing end of these galactans, regardless of their size, but with binding affinity increasing as the galactan chain length increases. Overall, our findings increase understanding as to how galactans interact with Gal-3 at the non-reducing, terminal end of galactose-containing polysaccharides as found on the cell surface.

Cloning, purification and biochemical characterisation of a GH35 beta-1,3/beta-1,6-galactosidase from the mucin-degrading gut bacterium Akkermansia muciniphila.

A putative GH35 β-galactosidase gene from the mucin-degrading bacterium Akkermansia muciniphila was successfully cloned and further investigated. The recombinant enzyme with the molecular mass of 74kDa was purified to homogeneity and biochemically characterised. The optimum temperature of the enzyme was 42°C, and the optimum pH was determined to be pH3.5. The addition of sodium dodecyl sulphate (SDS) reduced the enzyme's activity significantly. The addition of Mg2+-ions decreased the activity of the β-galactosidase, whereas other metal ions or EDTA showed no inhibitory effect. The enzyme catalysed the hydrolysis of β1,3- and β1,6- linked galactose residues from various substrates, whereas only negligible amounts of β1,4-galactose were hydrolysed. The present study describes the first functional characterisation of a β-galactosidase from this human gut symbiont.

Purification, molecular properties, structural characterization, and immunomodulatory activities of water soluble polysaccharides from Sargassum angustifolium

Sulfated polysaccharides isolated from Sargassum angustifolium and purified to determine their structural characteristics and biological activities. Crude polysaccharides and fractions (F1 and F2) were chiefly composed of neutral sugars (49.4–68.5%) and sulfates (12.5–23.0%) along with small amounts of uronic acids (1.3–13.6%) and proteins (4.1–4.7%). Polysaccharides were mainly constructed of different levels of fucose (23.9–69.9%) and galactose (22.5–29.8%) sugars. Subfractions with molecular weights ranging from 157.2 to 790.8×103g/mol were identified for isolated polysaccharides. Polysaccharides induced RAW264.7 macrophage cells to release noticeable amounts of nitric oxide and cytokines including IL-1β, TNF-α, IL-6, IL-10 and IL-12 through NF-κB and MAPKs signaling pathways. Sulfate esters of fraction F2 were necessary to its bioactivity and they were located on carbons 2, 4 and 6 of the major sugars. Fraction F2 was formed of (1→4)- and (1→3)-linked fucose residues branched at C-2 and C-4 as well as (1→6)-linked galactose residues branched at C-3.

Molecular glycopathology by capillary electrophoresis: Analysis of the N-glycome of formalin-fixed paraffin-embedded mouse tissue samples.

Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze endoglycosidase released and fluorophore-labeled N-glycans from formalin-fixed paraffin-embedded (FFPE) mouse tissue samples of lung, brain, heart, spleen, liver, kidney and intestine. The FFPE samples were first deparaffinized followed by solubilization and glycoprotein retrieval. PNGase F mediated release of the N-linked oligosaccharides was followed by labeling with aminopyrene trisulfonate. After CE-LIF glycoprofiling of the FFPE mouse tissues, the N-glycan pool of the lung specimen was subject to further investigation by exoglycosidase array based carbohydrate sequencing. Structural assignment of the oligosaccharides was accomplished by the help of the GUcal software and the associated database, based on the mobility shifts after treatments with the corresponding exoglycosidase reaction mixtures. Sixteen major N-linked carbohydrate structures were sequenced from the mouse lung FFPE tissue glycome and identified, as high mannose (3) neutral biantennary (3) sialylated monoantennary (1) and sialylated bianennary (9) oligosaccharides. Two of these latter ones also possessed alpha(1-3) linked galactose residues.

Characterization and immunomodulatory activities of polysaccharides extracted from green alga Chlorella ellipsoidea

Polysaccharides of green alga Chlorella ellipsoidea were extracted using hot water and fractionated using an anion-exchange chromatography to investigate their molecular characteristics and immunomodulatory activities. The crude polysaccharide and two fractions (F1 and F2) consisted mainly of carbohydrate (68.1–89.7%) and protein (2.0–11.8%) with small amounts of sulfate (1.9–6.1%) and uronic acid (0.5–6.1%). Glucose (58.8–97.6%) was the major monosaccharide of these polysaccharides, with different levels of rhamnose (0.2–11.6%), mannose (0.4–2.6%), and galactose (1.8–27.0%). The average molecular weights (Mw) of the crude, F1, and F2 were 175.8×103, 126.9×103, and 237.0×103g/mol, respectively. The crude, F1, and F2 stimulated murine macrophage, RAW264.7 cells, to produce considerable amounts of nitric oxide (NO) and various cytokines via up-regulation of their mRNA expression by the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. The fraction F2 with higher Mw and protein content showed stronger immunomodulatory activity. The main backbone of the fraction F2 was mainly connected via (1→4)-linked glucose and (1→6)-linked galactose with branches at C-3 and C-4 positions in (1→3,4)-linked glucose and (1→4,6)-linked galactose residues, respectively.

Further studies on structure of fucoidan from brown alga Saccharina gurjanovae.

A sulfated galactofucan SgF (MW 123kDa) was purified from the brown alga Saccharina gurjanovae. Polysaccharide was depolymerized by autohydrolysis at 25 and 60°C, and products were studied by mass spectrometry and (13)C NMR spectroscopy. According to results of investigation, the main chain of this polysaccharide is built of a repeating units →3)-α-L-Fucp-(2,4-OSO3(-))-(1→. Fucose chains could be sometimes terminated by (1→3)-linked galactose residues. Shorter (1→4)- and/or (1→6)-linked sulfated galactose chains are attached at positions C-2, C-3 of fucose residues. Sulfate groups can occupy positions C-2 and/or sometimes C-3 of Gal residues, but a sulfation at C-4 of the galactofucan could not be excluded. The SgF-AH25-H preparation (71kDa) was obtained by autohydrolysis of SgF at 25°C, which leaded to a selective desulfation at C-2 and, probably, to a cleavage of galactose chains, since structure of SgF-AH25-H represented a repeating unit →3)-α-l-Fucp-(4-OSO3(-))-(1→, which was definitely established by (13)C NMR spectroscopy. Galactofucan SgF and its derivative SgF-AH25-H exhibited no cytotoxic activity and leaded to about the same colony formation inhibition in colon cancer DLD-1 cells. Hence, structural simplification of SgF by lowering its molecular weight, desulfation at C-2 and removing of galactose residues by autohydrolysis at 25°C did not decrease its anticancer activity. This procedure allows obtaining standardized products which can be used as medical.

Chemical characteristics and anticoagulant activities of two sulfated polysaccharides from Enteromorpha linza (Chlorophyta)

Two sulfated polysaccharides, designated MP and SP, were extracted from the marine green alga Enteromorpha linza using hot water and then purified using ion-exchange and size-exclusion chromatography. The anticoagulant activities of MP and SP were examined by determination of their activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) using human plasma. Results showed that MP and SP were composed of abundant rhamnose with small amounts of xylose and glucuronic acid, whereas SP also contained a small amount of galactose. Approximate molecular weights of MP and SP were 535 and 502 kDa, respectively. As compared with SP, MP had higher contents of sulfate ester (19.0%) and uronic acid (14.9%). The MP mainly consisted of (1→4)-linked rhamnose residues with partially sulfated groups at the C-3 position, and small amounts of (1→3, 4)-linked rhamnose, (1→2, 4)-linked rhamnose, (1→4)-linked glucuronic acid and (1→4)-linked xylose residues. The SP contained abundant (1→4)-linked rhamnose with minor amounts of (1→3)-linked rhamnose, (1→3, 4)-linked rhamnose, (1→2, 4)-linked rhamnose, (1→4)-linked glucuronic acid, (1→4)-linked xylose, and (1→3)-linked galactose residues. The sulfate groups were mainly located at C-3 of (1→4)-linked rhamnose residues. Both MP and SP, in particular the former, effectively prolonged APTT and TT. This work demonstrates that MP and SP have unique structural characteristics distinct from those of other sulfated polysaccharides from Enteromorpha. The MP is a potential source of anticoagulant, and the difference in anticoagulant activities of the two sulfated polysaccharides is directly linked to the discrepancy of their chemical features.

ESIMS analysis of fucoidan preparations from Costaria costata, extracted from alga at different life-stages

Four fucoidan fractions from brown alga Costaria costata, collected at different life-stages: vegetative, May (5F2 and 5F3) and generative, July (7F1 and 7F2) collections were characterized. It was found that seaweed synthesizes different set of fucoidans – one with high fucose content and substantial percentage of hexoses and uronic acid and lower sulfate content (7F1, 5F2 and 5F3) and other – highly sulfated galactofucan (7F2). Structural features of fractions 7F2 and 5F3 were predominantly determined by mass spectrometric analysis of low-molecular-weight (LMW) oligosaccharide fragments, obtained by autohydrolysis of 7F2 and mild acid hydrolysis of 5F3 fucoidans. It was found that oligosaccharides from 7F2 fractions were mainly built up of sulfated at C-2 and/or at C-2/C-4 (1→3)-linked α-l-fucopyranose residues. d-Galactose residues, sulfated either at C-2 or C-6, were found as parts of mixed di- and trisaccharides at both termini and, probably, internal. Fucose residues in 5F3 fucoidan fragments were sulfated at C-2 and sometimes at C-4. Galactose residues were sulfated at C-4 and less frequently at C-2. Resistant to hydrolysis fraction was probably a core, built up with fucose, mannose and glucuronic acid. Presumably, oligosaccharide fragments were branches at C-4 of GlcA. They were sulfated at C-2 and sometimes at C-4 (1→3)- and/or (1→4)-linked fucooligosaccharides (sometimes terminated with (1→3)-linked galactose) and sulfated at C-4 or C-2 (1→4)- or, probably, (1→6)-linked galactooligosaccharides, probably, with own branches, formed by (1→2)-linked galactose residues. Unsulfated xylose residues were probably terminal in chains built up of fucose. It was confirmed, that monosaccharide content and structure of fucoidans of vegetative algae changed following its life stage. Generative alga in general produced highly sulfated galactofucan having lower MW along with less sulfated mannoglucuronofucan with higher MW, which was extensively synthesized by vegetative algae.

Sulfated Galactans of Champia indica and Champia parvula (Rhodymeniales, Rhodophyta) of Indian Waters

Sulfated galactans of the red seaweed species Champia indica and Champia parvula of Indian waters were extracted and purified by ion exchange chromatography. These were characterized by infrared and 13C NMR spectroscopy as well as by GC-MS analysis of alditol acetate derivatives produced by reductive hydrolysis/acetylation of sulfated and desulfated and their methylated samples. The sulfated galactans of these Champia species contained alternating β-(1→3)-linked galactopyranosyl units with sulfation at the 2-position and α-(1→4)-linked galactopyranosyl units having sulfation at both the 2- and 3-positions. Other minor substitutions included 6-O-methyl ether of the β-(1→3)-linked galactose residues only in Champia parvula.

Molecular Cloning and Characterization of a Novel β-Agarase, AgaB, from Marine Pseudoalteromonas sp. CY24

Agarases are generally classified into glycoside hydrolase families 16, 50, and 86 and are found to degrade agarose to frequently generate neoagarobiose, neoagarotetraose, or neoagarohexaose as the main products. In this study we have cloned a novel endo-type beta-agarase gene, agaB, from marine Pseudoalteromonas sp. CY24. The novel agarase encoded by agaB gene has no significant sequence similarity with any known proteins including all glycoside hydrolases. It degrades agarose to generate neoagarooctaose and neoagarodecaose as the main end products. Based on the analyses of enzymatic kinetics and degradation patterns of different oligosaccharides, the agarase AgaB appears to have a large substrate binding cleft that accommodates 12 sugar units, with 8 sugar units toward the reducing end spanning subsites +1 to +8 and 4 sugar units toward the non-reducing end spanning subsites -4 to -1, and enzymatic cleavage taking place between subsites -1 and +1. In addition, 1H NMR analysis shows that this enzyme hydrolyzes the glycosidic bond with inversion of anomeric configuration, in contrast to other known agarases that are retaining. Altogether, AgaB is structurally and functionally different from other known agarases and appears to represent a new family of glycoside hydrolase.

Structural features of pectins from fresh and sun-dried Japanese persimmon fruit

Softening-associated changes in a cold-water-soluble pectin, CWP3 of Japanese persimmon fruit are investigated by methylation analysis. Methylation analysis showed that rhamnose occurred as (1 → 2)- and (1 → 2,4)-linked residues in CWP3 (from fresh persimmon), newly solubilized CWP3 (fraction ‘a’ from sun-dried persimmon) and depolymerized CWP3 (fraction ‘b’ from sun-dried persimmon) as expected for the presence of ‘hairy’ regions. Solubilization of pectin into CWP3 occurred with higher proportions of (1 → 5)-linked arabinan side-chains and lower proportions of (1 → 3)/(1 → 6)-linked arabinogalactan side-chains compared to the respective side chains in CWP3 (fresh persimmon). During the sun-drying process, marked losses of (1 → 3,6)-linked galactose residues from CWP3 occurred, due to extensive degradation of main-chains of arabinogalactans. This implied losses of both types of sugars, arabinose (as terminal) and galactose from arabinogalactans in CWP3 of Japanese persimmon. During the sun-drying process, the molecular size distribution of CWP3 was affected due to degradation of arabinogalactan and arabinan side-chains.

The occurrence of internal (1→5)-linked arabinofuranose and arabinopyranose residues in arabinogalactan side chains from soybean pectic substances

CDTA-extractable soybean pectic substances were subjected to enzymatic digestion with arabinogalactan degrading enzymes yielding a resistant polymeric pectic backbone and arabino-, galacto-, and arabinogalacto-oligomers. The complex digest was fractionated using size-exclusion chromatography. Monosaccharide composition analysis, HPAEC fractionation and MALDI-TOF MS analysis of the resulting fractions showed that each contained a mixture of oligosaccharides of essentially the same degree of polymerisation, composed of only arabinose and galactose. MALDI-TOF MS analysis was used for molecular mass screening of oligosaccharides in underivatised HPAEC fractions. The monosaccharide sequence and the branching pattern of oligosaccharides (degree of polymerisation from 4 to 8) were determined using linkage analysis and ES-CID tandem MS analysis of the per-O-methylated oligosaccharides in each of the HPAEC fractions. These analyses indicated the presence of common linear (1→4)-linked galacto-oligosaccharides, and both linear and branched arabino-oligosaccharides. In addition, the results unambiguously showed the presence of oligosaccharides containing (1→4)-linked galactose residues bearing an arabino pyranose residue as the non-reducing terminal residue, and a mixture of linear oligosaccharides constructed of (1→4)-linked galactose residues interspersed with an internal (1→5)-linked arabino furanose residue. The consequences of these two new structural features of pectic arabinogalactan side chains are discussed.

Pectic substances from red beet ( Beta vulgaris conditiva). Part I. Structural analysis of rhamnogalacturonan I using enzymic degradation and methylation analysis

Cell wall material from ripe red beet ( Beta vulgaris L. var. conditiva) was isolated as alcohol insoluble residue (AIR). The chelator-soluble pectin obtained by cyclohexane- trans-1,2-diaminotetraacetate (CDTA) extraction of the AIR was fractionated by anion exchange chromatography (AEC). The main fraction was further fractionated by gel filtration chromatography (GFC). Fractions from both chromatographic systems were stepwise degraded by endo-polygalacturonase, endo-β-(1→4)- d-galactanase, endo-α-(1→5)- l-arabinanase and α- l-arabinofuranosidase. Degradation products were fractionated by GFC or by AEC. Polymeric fractions were investigated by methylation analysis after carbodiimide-activated reduction with NaBD 4. Selected fractions were additionally methylated with trideuteromethyliodide to enable the detection of O-methyl substituted sugars. The results indicate that the CDTA-soluble pectins of red beet cell walls are composed of three different sub-units: a homogalacturonan, which accounts for about 75%, a highly ramified rhamnogalacturonan I (RG-I) and a typical rhamnogalacturonan II (RG-II). RG-I consists of a highly ramified backbone composed of nearly equal amounts of rhamnose and galacturonic acid. Side chains, mainly arabinans, galactans and type-II arabinogalactans are attached to the RG-I backbone. Some arabinans are connected via short galactan chains directly or indirectly to this backbone. Type-II arabinogalactans are formed by “inner” chains consisting of (1→3)-linked galactans and short “outer” chains composed of an average number of one to three (1→6)-linked galactose residues. Terminal arabinofuranoses are linked via the O-3-position to galactose residues. Nearly all non-reducing ends consist of glucuronic acid. Approximately 65% of the glucuronic acid residues are substituted by a methyl ether group and approximately 10%, most probably, by a terminally linked rhamnose.

Purification and properties of recombinant beta-galactosidase from Bacillus circulans.

A gene encoding beta-galactosidase from Bacillus circulans which had hydrolysis specificity for the beta1-3 linkage was expressed in Escherichia coli. The beta-galactosidase was purified from crude cell lysates of E. coli by column chromatographies on Resource Q and Sephacryl S-200 HR. The enzyme released galactose with high selectivity from oligosaccharides which had terminal beta1-3 linked galactose residues. However it did not hydrolyse beta1-4 linked galactooligosaccharides. Moreover, Galbeta1-3GlcNAc, Galbeta1-3GalNAc, and their p-nitrophenyl glycosides were regioselectively synthesized in 10-46% yield by the transglycosylation reaction using this enzyme.

Biosynthesis of lipophosphoglycan from Leishmania major: characterization of (beta 1-3)-galactosyltransferase(s).

Lipophosphoglycan (LPG) is the major cell surface molecule of promastigotes of all Leishmania species. It is comprised of three domains: a conserved GPI anchor linked to a repeating phosphorylated disaccharide (P2; PO4-6-Gal(beta 1-4)Man(alpha 1-) backbone variously substituted with galactose, glucose and arabinose residues in L.major and capped with a neutral oligosaccharide. Using a microsomal membrane preparation from L.major, we have been able to demonstrate that galactose from UDP-[14C]galactose can be transferred to an endogenous acceptor, characterized as LPG. An in vitro assay was established, based on anion-exchange HPLC, that concurrently identifies and quantitates the products of the galactosyltransferases. We show that the products formed are [14C]galactose-labelled P3 (PO4-6-[Gal(beta 1-3)]Gal(beta 1-4)Man(alpha 1-), P4b (PO4-6-[Gal(beta 1-3)Gal(beta 1-3)]Gal(beta 1-4)Man(alpha 1-) and P5b(PO4-6-[Gal(beta 1-3)Gal(beta 1-3)Gal(beta 1-3)]Gal(beta 1- 4)Man(alpha 1-). These are major galactosylated repeating units of the backbone of L.major LPG. The same products are also formed when LPG from L.donovani, which contains an unbranched backbone of P2 repeats, is used as an exogenous acceptor with L.major microsomal membranes and UDP-[14C]galactose. In addition, no formation of radioactive backbone repeats (P2) was detected in membrane incubations containing UDP-[14C]galactose with or without added unlabelled GDP-mannose, indicating that the addition of the (beta 1-3)-linked galactose branches is independent of the synthesis of the repeating disaccharide (P2) backbone. Preliminary kinetic analyses suggest that the addition of multiple (beta 1-3)-linked galactose residues may be catalysed by more than one (beta 1-3) galactosyltransferase. The (beta 1-3)galactosyltransferase(s) activity was not detected in microsomal membrane preparations from promastigotes of L.donovani.

Structural characterization of the asparagine-linked oligosaccharides from Trypanosoma brucei type II and type III variant surface glycoproteins.

The complete primary structures of the major Asn-linked oligosaccharides from the type II variant surface glycoproteins (VSGs), MITat 1.2 and MITat 1.7, and the type III VSG, MITat 1.5, were determined using a combination of exo- and endoglycosidase digestions, methylation analysis, acetolysis, and 500 MHz 1H NMR spectroscopy. Each variant contained classical branched oligomannose-type and biantennary complex oligosaccharides, a proportion of the latter substituted with terminal alpha(1-3)-linked galactose residues, the first report of the presence of this epitope in Trypanosoma brucei. In addition both the type II variants contained relatively large amounts of the unusual small oligomannose-type oligosaccharides, Man4GlcNAc2 and Man3GlcNAc2, and a diverse array of novel branched poly-N-acetyllactosamine oligosaccharides, similar but not identical to those from mammalian glycoproteins. These latter structures were also partially substituted with terminal alpha(1-3)-linked galactose residues. Glycosylation in the type II variants showed site specificity in that the poly-N-acetyllactosamine and Man(9-5)GlcNAc2 oligosaccharides were located exclusively at Asn-glycosylation site 1 very close to the C terminus, whereas the Man(4-3)GlcNAc2 and biantennary complex oligosaccharides were located exclusively at site 2. This is the first report of the presence of poly-N-acetyllactosamine oligosaccharides in protozoa.

Structures of the alpha(1-3)-galactose-containing asparagine-linked glycans of a Lewis lung carcinoma cell subline resistant to Aleuria aurantia agglutinin: elucidation by 1H-NMR spectroscopy.

Four bi-antennary glycan fractions of the N-acetyllactosamine-type, derived from a Lewis lung carcinoma (LL2) cell subline resistant to the Aleuria aurantia agglutinin were studied by 400 MHz 1H-NMR spectroscopy. By this method, their antennae were found to be terminated either by alpha(2-3 or 6)-linked N-acetylneuraminic acid or alpha(1-3)-linked galactose residues. The primary structure of glycans of these four glycopeptide or derived oligosaccharide-alditols has been determined in full detail.

Structural features of cell-wall polysaccharides of potato ( Solanum tuberosum)

Cell-wall material from the parenchyma of mature potato tubers was fractionated by successive extractions with cyclohexane- trans-1,2-diaminetetra-acetate (CDTA) at 20°, 0.05 m Na 2CO 3 at 1° and 20°, 0.5 m and m KOH at 1°, m and 4 m KOH at 20°, and 4 m KOH + borate at 20° to leave the α-cellulose residue, which contained a significant amount of pectic material. The isolated polymers were fractionated by anion-exchange chromatography. The CDTA-soluble pectic polysaccharides were less branched than those solubilised by Na 2CO 3. The Na 2CO 3 (20°) extract contained appreciably more neutral side-chains. The residual cell wall contained cellulose and a highly branched pectic polysaccharide with a significant content of (1→4)-linked galactose residues. There were two main types of xyloglucan that had different degrees of branching. The less branched, solubilised by stronger alkali, were more strongly associated with the cellulose microfibrils. The cell wall contained 1.7% of protein and the content of hydroxyproline was low. Several of the fractions contained protein (0.2–18.0%), but only the fraction solubilised by 4 m KOH + borate contained a high level of hydroxyproline. The neutral component of this fraction was also rich in hydroxyproline and contained (1→2)- and (1→3)-linked arabinose.

Contact-dependent inhibition of growth of normal diploid human fibroblasts by plasma membrane glycoproteins

Homeotasis in vivo is maintained by a highly complex network of positive and negative signals. At the cellular level, this regulatory microenviroment can be divided, in a simplified fashion, into two major compartments: the humoral compartment, including compounds such as hormones, growth factors and nutrients, and the contact-environment compartment, including cell-cell and cell-matrix interactions. At least in cultures of diploid, non-transformed cells, cell-cell and cell-matrix interactions have been shown to be of major importance for the regulation of growth as well as of differentiation. Although until now the glycoprotein involved in the contact-dependent inhibition of growth has not been fully characterized, our studies give evidence for the involvement of a plasma membrane glycoprotein with an apparent molecular weight of ∼80 kDa in the growth regulation of diploid human fibroblast. The important characteristics of this glycoprotein is: the biologically active determinant resides in terminal, β-glycosidically linked galactose residues on N-glycosidically linked glycans. From our studies, a receptor has to be postulated which, in addition to the galactose residues, has additional structural requirements for the specific binding of this glycoprotein, sicne other glycoproteins carrying terminal, β-glycosidically linked galactose-residues are without biological activity. The postulated receptor is suggested to be defective in tumor cells, since these cells are no longer able to respond to cell-cell contacts with stopped proliferation, although they are able to inhibit growth of non-transformed cells. The inability of a tumor cell to recognize and to bind to the specific glycoprotein would result in a release from growth inhibition, leading to clonal growth of these cells. Further detailed studies on the structure and the regulation of the glycoprotein, as well as an attempt to isolate the postulated receptor, should lead to a better understanding of the complex pattern of growth regulation of normal cells.

Contact-dependent regulation of growth of diploid human fibroblasts is dependent upon the presence of terminal galactose residues on plasma membrane glycoproteins

The growth of diploid human fibroblasts has previously been shown to be regulated mainly by the extent of cell-cell contacts [ R. J. Wieser and F. Oesch (1986) J. Cell Biol. 103, 361], these contacts being effective only when terminal, β-glycosidically linked galactose residues were present on plasma membrane glycoproteins. These studies, in which a high cell density in sparse cell cultures has been mimicked by the addition of immobilized plasma membrane glycoproteins, have been further extended to investigate the role of terminal galactose residues directly in cell cultures. The studies presented herein show that (i) culturing human fibroblasts in the presence of β-galactosidase resulted in an approximately twofold higher saturation density, as well as a twofold higher proliferation rate at high cell densities when compared to the rates found in control cultures, (ii) The presence of α-lactalbumin in the culture medium, which acts as a modifier of the activity of galactosyltransferase, had the same effect as β-galactosidase. (iii) Addition of the lectin I from Bandeiraea simplicifolia (BS I), which is specific for terminal galactose residues, resulted in an increase in the proliferation rate of cell cultures at high cell densities, while the proliferation was not affected at low cell densities. These data show that the presence of terminal, β-glycosidically linked galactose is vital for the efficient growth control of normal cells.