Nectin-like molecule 1 is a glycoprotein with a single N-glycosylation site at N290KS which influences its adhesion activity

Nectin-like molecule 1 (NECL1)/CADM3/IGSF4B/TSLL1/SynCAM3, from now on referred to as NECL1, is a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule which has Ca²⁺-independent homo- or heterophilic cell-cell adhesion activity and plays an important role in the formation of synapses, axon bundles and myelinated axons. Here we first detected the expression of NECL1 in human fetal and adult brains, and mouse brains at different developmental stages. The results indicated that two bands with molecular weights of about 62 kDa and 48 kDa were found in human fetal brain, while only one band with a molecular weight of about 48 kDa was found in human adult brain; two bands with molecular weights of about 62 kDa and 48 kDa whose expression level gradually increased were also found from mouse E16 to P14, while only one band with a molecular weight of about 48 kDa was found from P14. Bioinformatics analysis showed there were two putative N-glycosylation sites within human NECL1 at positions N25LS and N290KS and within mouse Necl1 at positions N23LS and N288KS, respectively. There was no O-glycosylation site in either human NECL1 or mouse Necl1. Based on the results of N-Glycosidase F treatment with human fetal brain tissue and lysates from transient transfection with human wild-type or glycosylation site mutant NECL1 in 293ET cells, we demonstrated that human NECL1 is an N-linked glycoprotein with a single glycosylation site at position N290KS. Cell aggregation assay further showed there was an increased adhesion activity after the glycosylation site mutation of NECL1 molecule.     

Nectin-like molecule 1 is a protein 4.1N associated protein and recruits protein 4.1N from cytoplasm to the plasma membrane

Nectins are immunoglobulin superfamily adhesion molecules that participate in the organization of epithelial and endothelial junctions. Sharing high homology with the poliovirus receptor (PVR/CD155), nectins were also named poliovirus receptor-related proteins (PRRs). Four nectins and five nectin-like molecules have been identified. Here we describe the cloning and characterization of human and mouse nectin-like molecular 1 (NECL1). Human and mouse NECL1 share 87.3% identity at the amino acid level. NECL1 contains an ectodomain made of three immunoglobulin-like domains, and a cytoplasmic region homologous to those of glycophorin C and contactin-associated protein. RNA blot and in situ hybridization analysis showed that NECL1 predominantly expressed in the central nervous system, mainly in neuronal cell bodies in a variety of brain regions including the cerebellum, cerebral cortex and hippocampus. In vitro binding assay proved the association of NECL1 with protein 4.1N. NECL1 localizes to the cell-cell junctions and recruits protein 4.1N to the plasma membranes through its C-terminus, thus may regulate the function of the cell-cell junction. We propose that the NECL1 and protein 4.1N complex is involved in the morphological development, stability, and dynamic plasticity of the nervous system.     

Nectin-like molecule 1 is a protein 4.1N associated protein and recruits protein 4.1N from cytoplasm to the plasma membrane

Nectins are immunoglobulin superfamily adhesion molecules that participate in the organization of epithelial and endothelial junctions. Sharing high homology with the poliovirus receptor (PVR/CD155), nectins were also named poliovirus receptor-related proteins (PRRs). Four nectins and five nectin-like molecules have been identified. Here we describe the cloning and characterization of human and mouse nectin-like molecular 1 (NECL1). Human and mouse NECL1 share 87.3% identity at the amino acid level. NECL1 contains an ectodomain made of three immunoglobulin-like domains, and a cytoplasmic region homologous to those of glycophorin C and contactin-associated protein. RNA blot and in situ hybridization analysis showed that NECL1 predominantly expressed in the central nervous system, mainly in neuronal cell bodies in a variety of brain regions including the cerebellum, cerebral cortex and hippocampus. In vitro binding assay proved the association of NECL1 with protein 4.1N. NECL1 localizes to the cell-cell junctions and recruits protein 4.1N to the plasma membranes through its C-terminus, thus may regulate the function of the cell-cell junction. We propose that the NECL1 and protein 4.1N complex is involved in the morphological development, stability, and dynamic plasticity of the nervous system.     

Nectin-like protein 2 defines a subset of T-cell zone dendritic cells and is a ligand for class-I-restricted T-cell-associated molecule

Dendritic cells (DCs) are a phenotypically and functionally heterogenous population of leukocytes with distinct subsets serving a different set of specialized immune functions. Here we applied an in vitro whole cell panning approach using antibody phage display technology to identify cell-surface epitopes specifically expressed on human blood BDCA3(+) DCs. A single-chain antibody fragment (anti-1F12 scFv) was isolated that recognizes a conserved surface antigen expressed on both human BDCA3(+) DCs and mouse CD8alpha(+) DCs. We demonstrate that anti-1F12 scFv binds Nectin-like protein 2 (Necl2, Tslc1, SynCaM, SgIGSF, or Igsf4), an adhesion molecule involved in tumor suppression, synapse formation, and spermatogenesis. Thus, Necl2 defines a specialized subset of DCs in both mouse and human. We further show that Necl2 binds Class-I-restricted T-cell-associated molecule (CRTAM), a receptor primarily expressed on activated cytotoxic lymphocytes. When present on antigen presenting cells, Necl2 regulates IL-22 expression by activated CD8(+) T-cells. We propose that Necl2/CRTAM molecular pair could regulate a large panel of cell/cell interactions both within and outside of the immune system.     

The Distribution and Biochemical Properties of a Cdc2-Related Kinase, KKIALRE, in Normal and Alzheimer Brains

Abstract: The biochemical properties and distribution of a Cdc2-related kinase, KKIALRE, were studied in brain tissues and cultured cells with antibodies to a subregion of KKIALRE protein deduced from cDNA. In adult human brain, the KKIALRE-immunoreactive protein consisted of four or five isoforms having a molecular size of 40–52 kDa, whereas in fetal brain, there was one protein of ∼48 kDa. Cultured astrocytes, neuroblastoma cells, and mouse brains contained the fetal form of KKIALRE protein. KKIALRE-immunoreactive proteins were capable of phosphorylating histone and synthetic peptides with the X-Ser-Pro-X motif, indicating that these proteins belong to the proline-directed Ser/Thr protein kinase family. The KKIALRE immunoreactivity was detected primarily in fibrous astrocytes in white matter and perivascular and subpial spaces, as well as in Bergmann glia in the cerebellum. In fetal brains radial glia were weakly immunoreactive. Reactive astrocytes were more intensely labeled than other glia. Neurons in normal brains and brains with Alzheimer's disease (AD) displayed no KKIALRE immunoreactivity. KKIALRE immunoreactivity was similar in neurons with and without neurofibrillary tangles. The results indicate that in CNS, the KKIALRE protein is mainly a glial protein that is up-regulated in gliosis and that it probably plays no role in the hyperphosphorylation of τ in AD brains.     

Expression of galectins on microvessel endothelial cells and their involvement in tumour cell adhesion

Lactoside-binding lectins (galectins) with molecular weights of about 14.5 kDa (galectin-1) and 29–35 kDa (galectin-3) bind preferentially to polylactosaminoglycan-containing glycoconjugates and have been found on the surface of tumour cells and implicated in cell-cell and cell-extracellular matrix adhesion and metastasis. We have demonstrated by immunoblotting that both galectin-1 and galectin-3 are present in extracts of endothelial cells cultured from bovine aorta, rat lung, mouse lung and mouse brain microvessels, whereas mouse hepatic sinusoidal endothelial cells expressed primarily galectin-1. These galectins were also localized by indirect immunofluorescent labelling on the surface of the different endothelial cells in culture and by immunohistochemical staining in human tissuesin vivo. Anti-galectin-1 antibodies inhibited the adhesion of liver-preferring murine RAW117-H10 large-cell lymphoma cells to hepatic sinusoidal endothelial cells or lung microvessel endothelial cellsin vitro. The data indicate that galectin-1 is expressed on the extracellular surface of endothelial cells and can mediate in part the adhesion of RAW117-H10 cells to liver microvessel endothelial cells.     

Identification of a novel beta-catenin-interacting protein

Cadherin is a well-known cell-cell adhesion molecule, and it binds to beta-catenin, which in turn binds to alpha-catenin. However, little is known about the regulatory mechanism underlying the cadherin-mediated cell-cell adhesion. Here we purified two novel beta-catenin-interacting proteins with molecular masses of 180 kDa (p180) and 150 kDa (p150) from bovine brain cytosol by using glutathione S-transferase (GST)-beta-catenin affinity column chromatography. Mass spectral analysis revealed p180 to be identical to KIAA0313 which has a putative Rap1 guanine nucleotide exchange factor (GEF) domain and p150 to be the same as KIAA0705 which has a high degree of sequence similarity to the synaptic scaffolding molecule (S-SCAM), which binds beta-catenin and KIAA0313 in the yeast two-hybrid system and overlay assay, respectively (Ide et al., Biochem. Biophys. Res. Commun. 256, 456-461, 1999; Ohtsuka et al., Biochem. Biophys. Res. Commun. 265, 38-44, 1999). beta-Catenin was coimmunoprecipitated with KIAA0313 in Madin-Darby canine kidney II (MDCKII) cells, bovine brain cytosol, and EL cells. KIAA0313 and beta-catenin were partly colocalized at sites of cell-cell contact in MDCKII cells. Taken together, our data suggest that KIAA0313 associates with beta-catenin through KIAA0705 in vivo at sites of cell-cell contact.     

Cloning and characterization of a lysosomal phospholipase A2, 1-O-acylceramide synthase.

Recently, a novel enzyme, 1-O-acylceramide synthase (ACS), was purified and characterized from bovine brain. This enzyme has both calcium-independent phospholipase A(2) and transacylase activities. The discovery of this enzyme led us to propose a new pathway for ceramide metabolism in which the sn-2-acyl group of either phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl group of ceramide. In this study, the partial amino acid sequences from the purified enzyme revealed that the enzyme contains amino acid sequences identical to those of human lecithin:cholesterol acyltransferase-like lysophospholipase (LLPL). The coding sequences of the mouse, bovine, and human genes were obtained from the respective kidney cDNAs by PCR. The open reading frames of LLPL were cloned into pcDNA3 to generate carboxyl-terminally tagged proteins. The expression of mouse LLPL in COS-7 cells demonstrated that transfected cells had higher transacylase and phospholipase A(2) activities than did non-transfected cells. Immunoprecipitation confirmed that LLPL had ACS activity. There were no significant lecithin:cholesterol acyltransferase and lysophospholipase activities in the mouse LLPL-transfected cells under either acidic or neutral conditions. Amino acid sequences from cDNAs of mouse, human, and bovine LLPLs demonstrated a signal peptide cleavage site, one lipase motif (AXSXG), and several N-linked glycosylation sites in each LLPL molecule. The replacement of serine with alanine in the lipase motif of mouse LLPL resulted in elimination of enzyme activity, indicating that the serine residue is part of the catalytic site. Deglycosylation of mouse, human, and bovine LLPLs yielded core proteins with a molecular mass of 42 kDa without change in enzyme activities. LLPL was post-translationally modified by signal peptide cleavage and N-linked glycosylation, and each mature LLPL had the same size core protein. Subcellular fractionation demonstrated that ACS activity co-localized with N-acetylglucosaminidase. Therefore, LLPL encodes a novel lysosomal enzyme, ACS.     

Alcohol inhibits cell-cell adhesion mediated by human L1 [published erratum appears in J Cell Biol 1996 Jun;133(5):1139-40]

Mental retardation, hydrocephalus, and agenesis of the corpus callosum are observed both in fetal alcohol syndrome (FAS) and in children with mutations in the gene for the cell adhesion molecule L1. We studied the effects of ethanol on cell-cell adhesion in mouse fibroblasts transfected with human L1. L1-transfected fibroblasts exhibited increased cell-cell adhesion compared with wild-type or vector- transfected controls. Ethanol potently and completely inhibited L1- mediated adhesion both in transfected L cells and NIH/3T3 cells. Half- maximal inhibition was observed at 7 mM ethanol, a concentration achieved in blood and brain after ingesting one alcoholic beverage. In contrast, ethanol did not inhibit the adhesion of fibroblasts transfected with vector alone or with N-CAM-140. L1-mediated cell-cell adhesion was inhibited with increasing potency by n-propanol and n- butanol, but was not inhibited at all by n-alcohols of 5 to 8 carbons, acetaldehyde, or acetate, suggesting that ethanol interacts directly with a small hydrophobic pocket within L1. Phenylalanine, teratogenic anticonvulsants, and high concentrations of glucose did not inhibit L1- mediated cell-cell adhesion. Ethanol also inhibited potently the heterotypic adhesion of rat cerebellar granule cells to a monolayer of L1-transfected NIH/3T3 cells, but had no effect on their adhesion to N- CAM-140 or vector-transfected NIH/3T3 cells. Because L1 plays a role in both neural development and learning, ethanol inhibition of L1-mediated cell-cell interactions could contribute to FAS and ethanol-associated memory disorders.     

Oligosaccharide-Related Epitope Specific for a Brain-Specific Glycoprotein, 1D4 Antigen

The characteristics of glycosylation of a brain-specific glycoprotein, 1D4 antigen, and the epitope recognized by its monoclonal antibody were studied. Removal of high-mannose and hybrid types of N-linked oligosaccharides by treatment with endoglycosidase H converted the molecular mass of the 1D4 antigen from 89 kDa to 78 kDa, but did not affect its reactivity with the 1D4 monoclonal antibody. Removal of all types of N-linked oligosaccharides by treatment with glycopeptidase F or removal of both N- and O-linked oligosaccharides by chemical treatment caused both reduction of the molecular mass of the antigen to 63 kDa and loss of its reactivity with the monoclonal antibody. These results suggest that the 1D4 monoclonal antibody recognizes a complex-type oligosaccharide-related epitope specific for the 1D4 antigen. Results also showed that N-linked glycosylation was not responsible for the charge heterogeneity of the 1D4 antigen. The oligosaccharide chain-related epitope was detected in rat brain but not in mouse, rabbit, or bovine brain, but the 1D4 antigen was recognized in rat and mouse brains with antiserum (polyclonal antibodies). These findings indicate that the oligosaccharide-related epitope is species specific. Furthermore, results with neuraminidase-treated 1D4 antigen indicated that sialic acids were not involved in the oligosaccharide-related epitope. These findings suggest that the 1D4 antigen may have the oligosaccharide structure specific for rat brain and itself.     

Lack of protein 4.1G causes altered expression and localization of the cell adhesion molecule nectin-like 4 in testis and can cause male infertility

Protein 4.1G is a member of the protein 4.1 family, which in general serves as adaptors linking transmembrane proteins to the cytoskeleton. 4.1G is thought to be widely expressed in many cells and tissues, but its function remains largely unknown. To explore the function of 4.1G in vivo, we generated 4.1G(-/-) mice and bred the mice in two backgrounds: C57BL/6 (B6) and 129/Sv (129) hybrids (B6-129) and inbred B6. Although the B6 4.1G(-/-) mice showed no obvious abnormalities, deficiency of 4.1G in B6-129 hybrids was associated with male infertility. Histological examinations of these 4.1G(-/-) mice revealed atrophy, impaired cell-cell contact and sloughing off of spermatogenic cells in seminiferous epithelium, and lack of mature spermatids in the epididymis. Ultrastructural examination revealed enlarged intercellular spaces between spermatogenic and Sertoli cells as well as the spermatid deformities. At the molecular level, 4.1G is associated with the nectin-like 4 (NECL4) adhesion molecule. Importantly, the expression of NECL4 was decreased, and the localization of NECL4 was altered in 4.1G(-/-) testis. Thus, our findings imply that 4.1G plays a role in spermatogenesis by mediating cell-cell adhesion between spermatogenic and Sertoli cells through its interaction with NECL4 on Sertoli cells. Additionally, the finding that infertility is present in B6-129 but not on the B6 background suggests the presence of a major modifier gene(s) that influences 4.1G function and is associated with male infertility.     

Isolation and characterization of a membrane glycoprotein from human brain with sequence similarities to cell adhesion proteins from chicken and mouse

We previously described the production of monoclonal antibodies against a preparation of membrane glycoproteins from human brain [Berglund et al. (1987) J. Neurochem. 48, 809-815]. One of the glycoproteins, recognized by monoclonal antibody CF3, was specifically expressed in the brain. We now report the isolation and characterization of this glycoprotein, called glycoprotein 135 (Gp135). Gp135 was purified by means of lentil lectin affinity chromatography and immunoaffinity chromatography, using monoclonal antibody CF3, from a crude membrane extract of human brain cortex. Gp135 was shown to consist of a glycosylated single polypeptide chain with an apparent molecular mass of 135 kDa. The size of the polypeptide moiety was estimated to 115 kDa following N-glycanase digestion. The glycoprotein is anchored in the membrane by a glycosylphosphatidylinositol tail, as shown by phospholipase C digestion and liposome incorporation experiments. Amino acid sequence analysis of the amino terminal, and of an internal peptide obtained by V8 protease digestion of the glycoprotein, revealed a strong similarity to three previously described glycoproteins from chicken (contactin and F11) and mouse (F3) brains. These glycoproteins belong to the immunoglobulin superfamily and are implicated in cell adhesion phenomena in the developing brain. Gp135 may be the human counterpart to one or several of these glycoproteins.     

Molecular cloning and expression of human neurochondrin-1 and -2.

Human neurochondrins have been cloned from a brain cDNA library. The human neurochondrin-1 and -2 predict leucine-rich (15.8 and 15.9%) proteins of 729 and 712 amino acid residues, with molecular weights of 78.9 and 77.2 kDa, respectively. The deduced amino acid sequence indicates 98% identity among human, mouse and rat species. Northern analysis indicates that about 4 kb human neurochondrin mRNAs are abundant in the fetal and the adult brain.     

Characterization of the biosynthesis,membrane association and function of the cell adhesion molecule L1

The L1 cell adhesion molecule is involved in cell migration and cell-cell adhesion in the brain. In this report we describe a purification procedure which allowed simultaneous isolation of L1 and the neural cell adhesion molecule. Furthermore, we studied L1 biosynthesis, post-translational modifications and function. L1 was synthesized as a polypeptide with relative molecular weight 200,000 in monolayer cultures of fetal rat neurons and in explant cultures of rat forebrain. The L1 polypeptide was co-translationally inserted into the membranes of the endoplasmic reticulum as an integral membrane protein. Both sulfation and phosphorylation of L1 was observed. L1 polypeptides with lower relative molecular weight which were present on the plasma membrane were probably derived from the 200,000 mol. wt polypeptide. The function of L1 was investigated and an L1 involvement in neurite fasciculation was demonstrated.     

Molecular cloning of a human Ca2+-dependent cell-cell adhesion molecule homologous to mouse placental cadherin: its low expression in human placental tissues

P-cadherin is a subclass of Ca2+-dependent cell-cell adhesion molecules present in mouse placenta, where its localization suggests a function of connecting the embryo to the uterus (Nose, A., and M. Takeichi. 1986. J. Cell Biol. 103:2649-2658). We recently identified a human cadherin detected by an mAb capable of disrupting cell-cell adhesion of A-431 cells, and found that it was closely related immunochemically to mouse P-cadherin. Curiously, this cadherin was undetectable in human placenta by immunohistochemical examination (Shimoyama, Y., S. Hirohashi, S. Hirano, M. Noguchi, Y. Shimosato, M. Takeichi, and O. Abe. 1989. Cancer Res. 49:2128-2133). We here report the cloning and sequencing of cDNA clone encoding the human homologue of mouse P- cadherin. The deduced amino acid sequence of the human P-cadherin consists of 829 amino acid and shows striking homology with mouse P- cadherin. On Northern blot analysis, human P-cadherin was scarcely expressed in human placenta in contrast to mouse P-cadherin, which was abundantly expressed in mouse placenta throughout pregnancy, and it was shown that E-cadherin, but not P-cadherin, was the major cadherin molecule in human placenta. Moreover, NIH3T3 cells transfected with human P-cadherin cDNA expressed the functional cadherin molecule, which was identical to the cadherin we had previously identified using the mAb, showing that this molecule really does mediate cell-cell adhesion and that the cadherin we detected immunochemically is undoubtedly human P-cadherin. The results obtained in this study support the idea that P- cadherin plays little role, if any, in Ca2+-dependent cell-cell binding in human placental tissue at least after several weeks of pregnancy.     

Sphingomyelinase activities in neuronal cell cultures.

Human and rat brains have been previously demonstrated to contain three sphinomyelinases, one lysosomal with a pH optimum of 5.0, one with a pH optimum of 7.4 and a dependence on magnesium and another with a pH optimum of 7.0 and no divalent cation requirement. Using brain cell cultures and clonal cell lines of both neuronal and glial origin the activities of the pH 5.0 and pH 7.4 (magnesium-dependent) sphingomyelinase were examined. Sphingomyelinase activity measured at pH 5.0 was found in all the cell lines tested including G26, C6, N18 (differentiated and undifferentiated), mouse “L” cells, human skin fibroblasts, fetal mouse brain surface cultures and fetal mouse brain reaggregate cultures. However, pH 7.4 (magnesium-dependent) sphingomyelinase activity was found only in the N18 cell lines and the reaggregate cultures suggesting the probable localization of this activity in neurons. Although the pH 7.4 sphingomyelinase activity was found in the fetal mouse brain used for the surface cultures this activity was rapidly lost. This enzyme may play an important role in neuronal development and maturation.     

Is PECAM-1 a mechanoresponsive molecule?

Endothelial cells are capable of responding to fluid shear stress, but the molecular mechanism for this biological response remains largely unknown. Our studies indicate that the cell-cell adhesion site is a possible site of flow sensing. PECAM-1, a cell adhesion molecule localized to the interendothelial cell adhesion site, is tyrosine-phosphorylated when endothelial cells are exposed to physiological levels of fluid shear stress. This PE-CAM-1 phosphorylation initiates a signaling cascade leading to ERK activation. Here we review what is known about PECAM-1 tyrosine phosphorylation and suggest a possible role of PECAM-1 in mechanosensing by endothelial cells.     

Molecular cloning, expression, and chromosomal localization of a human tubulointerstitial nephritis antigen

Tubulointerstitial nephritis antigen (TIN-ag) is an extracellular matrix basement protein which was originally identified as a target antigen involved in anti-tubular basement membrane (TBM) antibody-mediated interstitial nephritis (TIN). Further investigations elucidated that TIN-ag plays a role in renal tubulogenesis and that TIN-ag is defected in hereditary tubulointerstitial disorder such as juvenile nephronophthisis. We previously isolated and characterized 54 kDa glycoprotein as TIN-ag. cDNA encoding rabbit and mouse TIN-ag has recently been identified. In the present study, the cDNA of the human homologue of TIN-ag was cloned and its nucleotide sequence was determined (Accession No. AB022277; the DDBJ nucleotide sequence database). Deduced amino acid sequence (476 aa) exhibited the presence of a signal peptide (1-18 aa), cysteine residues termed follistatin module, six potential glycosylation sites, and an ATP/GTP-binding site. Homology search revealed approximately 85% homology with both rabbit and mouse TIN-ag, and also some ( approximately 40%) similarity with C. elegans. Human TIN-ag contained a sequence similar to several classes of extracellular matrix molecules in amino terminal region and to cathepsin family of cysteine proteinases in the carboxyl terminal region. Northern blot analysis revealed exclusive expression of this molecule in human adult and fetal kidney tissues. Using a monoclonal antibody recognizing human TIN-ag, protein expression ( approximately 50 kDa) was identified in cultured COS-1 cells transfected with human TIN-ag cDNA. The human TIN-ag was mapped to chromosome 6p11.2-12 by fluorescence in situ hybridization. These results may provide further evidence for understanding TIN-ag molecule and clues for gene analysis of juvenile nephronophthisis.     

Glycosylation of procathepsin L does not account for species molecular-mass differences and is not required for proteolytic activity.

Cathepsin L is a major lysosomal cysteine proteinase in mouse and human cells. Despite similar predicted molecular masses, procathepsin L in these two species migrates on SDS/polyacrylamide gels with apparent molecular masses of 39 kDa and 42 kDa respectively. To determine if glycosylation differences account for this discrepancy, and to ascertain whether glycosylation is essential for enzymic activity, mouse and human procathepsins L were expressed at high concentrations in mouse NIH 3T3 cells or in human A431 cells after DNA-mediated transfection of cloned DNAs for these enzymes. In pulse-chase studies, human procathepsin L transfectants synthesized and secreted large amounts of enzymically active 42 kDa proenzyme and processed it into 34 kDa and 26 kDa intracellular peptides, a pattern of secretion and processing similar to that seen with endogenous or transfected mouse procathepsin L. Both translation of cloned procathepsin L cDNAs in vitro and Endoglycosidase H treatment of 39 kDa mouse and 42 kDa human procathepsin L resulted in non-glycosylated proteins 2 kDa lower in molecular mass than the untreated proteins for both species. This suggests that glycosylation differences are not responsible for the molecular-mass disparity between the two species. Moreover, Endoglycosidase H-treated mouse enzyme retained full proteolytic activity, indicating that glycosylation of cathepsin L is not essential for enzymic function.     

Effect of lengthening lymphocyte function-associated antigen 3 on adhesion to CD2.

The effect of lengthening the distance in an adhesion molecule between the receptor binding site and the membrane anchor was studied by inserting four Ig-like domains into the two Ig domain lymphocyte function-associated antigen 3 (LFA-3) molecule. The extended molecule expressed in Chinese hamster ovary (CHO) cells bound to CD2 on T lymphocytes 4- to 20-fold more efficiently than the wild-type molecule at 4 degrees C. Treatment of the CHO clones with neuraminidase to remove sialic acid, or with deoxymannojirimycin to reduce the bulk of N-linked glycosylation, showed that adhesion to both the wild-type and the chimeric LFA-3 molecules was under the influence of cell-cell repulsive forces to a similar extent and that these treatments had less effect than lengthening LFA-3. At higher temperatures, such as 22 and 37 degrees C, the efficiency of binding to the wild-type LFA-3 increased to levels comparable with binding to extended LFA-3. Our results suggest that more distal locations of the adhesive binding site from the cell membrane anchor increase the efficiency of cell-cell adhesion by enhancing the frequency of receptor encounter with ligand and that more proximal locations of the adhesive binding site can provide efficient cell-cell adhesion at physiological temperatures.