Mouse "protective protein". cDNA cloning, sequence comparison, and expression

The "protective protein" is the glycoprotein that forms a complex with the lysosomal enzymes beta-galactosidase and neuraminidase. Its deficiency in man leads to the metabolic storage disorder galactosialidosis. The primary structure of human protective protein, deduced from its cloned cDNA, shows homology to yeast serine carboxypeptidases. We have isolated a full-length cDNA encoding murine protective protein. The nucleotide sequences as well as the predicted amino acid sequences are highly conserved between man and mouse. Domains important for the protease function are completely identical in the two proteins. Both human and mouse mature protective proteins covalently bind radiolabeled diisopropyl fluorophosphate. Transient expression of the murine cDNA in COS-1 cells yields a protective protein precursor of 54 kDa, a size characteristic of the glycosylated form. This cDNA-encoded precursor, endocytosed by human galactosialidosis fibroblasts, is processed into a 32- and a 20-kDa heterodimer and corrects beta-galactosidase and neuraminidase activities. A tissue-specific expression of protective protein mRNA is observed when total RNA from different mouse organs is analyzed on Northern blots.     

Expression of cDNA encoding the human "protective protein" associated with lysosomal beta-galactosidase and neuraminidase: homology to yeast proteases

The "protective protein" is a glycoprotein that associates with lysosomal beta-galactosidase and neuraminidase and is deficient in the autosomal recessive disorder galactosialidosis. We have isolated the cDNA encoding human "protective protein". The clone recognizes a 2 kb mRNA in normal cells that is not evident in fibroblasts of an early infantile galactosialidosis patient. The cDNA directs the synthesis of a 452 amino acid precursor molecule that is processed in vivo to yield mature "protective protein," a heterodimer of 32 kd and 20 kd polypeptides held together by disulfide bridges. This mature form is also biologically functional since it restores beta-galactosidase and neuraminidase activities in galactosialidosis cells. The predicted amino acid sequence of the "protective protein" bears homology to yeast carboxypeptidase Y and the KEX1 gene product. This suggests a protease activity for the "protective protein."     

The relation between human lysosomal beta-galactosidase and its protective protein

Cultured skin fibroblasts from patients with the lysosomal storage disease galactosialidosis lack a 54-kDa protein which is a precursor of 32-kDa and 20-kDa proteins, which immunoprecipitate with human anti-beta-galactosidase antiserum. The lack of a 32-kDa "protective protein" results in a combined deficiency of beta-galactosidase and sialidase. The mechanism of protection of lysosomal beta-galactosidase against proteolytic degradation is elucidated by sucrose density gradient centrifugation and immunoprecipitation studies. In normal fibroblasts at the low intralysosomal pH, more than 85% of beta-galactosidase exists as a high molecular weight (600-700 kDa) multimer and about 10% as a monomer of 64-kDa. In mutant cells from galactosialidosis patients, the residual enzyme activity, about 10%, is present as a monomer and no multimer exists. After addition of the 54-kDa precursor form of the protective protein, the density pattern of beta-galactosidase in galactosialidosis cells is normalized. Immunoprecipitation studies after sucrose density gradient centrifugation on homogenate and on purified beta-galactosidase from normal fibroblasts show that the protective protein is associated only with the multimeric form of beta-galactosidase. We propose that intralysosomal protection against proteolysis of beta-galactosidase and sialidase is accomplished by aggregation into a high molecular weight complex consisting of multimeric beta-galactosidase, sialidase, and protective protein. The genetic deficiency of the latter, as in galactosialidosis, results in a rapid degradation of monomeric beta-galactosidase and a loss of sialidase activity.     

Immunoelectron microscopical localization of lysosomal beta-galactosidase and its precursor forms in normal and mutant human fibroblasts

Immunoelectron microscopy was performed to study the biosynthesis of lysosomal beta-galactosidase (beta-gal) in normal and mutant human fibroblasts. Using polyclonal and monoclonal antibodies we show in normal cells precursor forms of beta-gal in the rough endoplasmic reticulum (RER) and in the Golgi apparatus throughout the stack of cisternae. In the lysosomes virtually all beta-gal exists as a high molecular weight multimer of mature enzyme. In the autosomal recessive disease GM1-gangliosidosis caused by a beta-gal deficiency and in galactosialidosis, associated with a combined deficiency of lysosomal neuraminidase and beta-gal, precursor forms of the latter enzyme are found in RER, Golgi and some labeling is present at the cell surface. The lysosomes remain unlabeled, indicative for the absence of enzyme molecules in this organelle. In galactosialidosis fibroblasts also no mature beta-gal is found in the lysosomes but in these cells the presence of the monomeric form can be increased by leupeptin (inhibition of proteolysis) whereas addition of a partly purified 32 kDa "protective protein" results in the restoration of high molecular weight beta-gal multimers in the lysosomes.     

Deficient lysosomal carboxypeptidase activity in galactosialidosis

In the lysosome, the glycosidases neuraminidase (EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23) are associated to a 52 kDa "protective protein" to form a large multi-enzymatic complex. Deficient synthesis or inactivation of this protective protein causes galactosialidosis, a lysosomal storage disorder in man in which both neuraminidase and beta-galactosidase activities are deficient. Since the protective protein possesses extensive sequence homology with carboxypeptidase Y (carb Y) and the KEX 1 gene product from yeast, we have used the artificial substrate N-CBZ-Phe-Leu to detect and characterize the peptidase activity of the lysosomal carboxypeptidase (carb L). Using both a purified preparation of the lysosomal multi-enzymatic complex and cultured skin fibroblasts of patients affected with galactosialidosis, we demonstrate that the 52 kDa protective protein is responsible for carb L activity. The fibroblasts of three patients affected with late infantile and juvenile galactosialidosis were found to be deficient in carb L activity (1.4% of normal mean value).     

Molecular heterogeneity in human beta-galactosidase and neuraminidase deficiency

Human lysosomal beta-galactosidase and neuraminidase exist in a complex together with a 32-kilodalton (kd) glycoprotein. The latter protein was found to have a dual function: it is required for the aggregation of monomeric 64-kd beta-galactosidase into high molecular weight (600-700 kd) multimers and it is an essential subunit of neuraminidase together with a 76-kd polypeptide. The severe neurological disorder galactosialidosis, characterized by a coexistent deficiency of beta-galactosidase and neuraminidase, was found to be due to a genetic defect of the 32-kd protective protein. The molecular background of the clinical heterogeneity within this syndrome is described and will undoubtedly be further elucidated since we have recently isolated the gene coding for the protective protein. The sequence of normal and mutant (enzyme) proteins will also provide better insight into the characteristics of the beta-galactosidase-neuraminidase-protective protein complex. Another interesting model for the study of posttranslational processing is the defective phosphorylation of beta-galactosidase in cells from patients with GM1-gangliosidosis.     

Galactosialidosis: simultaneous deficiency of esterase, carboxy-terminal deamidase and acid carboxypeptidase activities

Esterase and deamidase activities at pH 7.0 and carboxypeptidase activity at pH 5.7 were markedly low or deficient in seven galactosialidosis fibroblast strains with deficient activity of "protective protein" for lysosomal beta-galactosidase and neuraminidase. No simultaneous deficiency of these three enzyme activities was observed in other lysosomal disease fibroblasts examined in this study. This result strongly suggests that "protective protein" is identical with a multifunctional protein with esterase/deamidase/carboxypeptidase activities and its mutation in galactosialidosis results in deficiency of these three enzyme activities.     

Transport of human lysosomal neuraminidase to mature lysosomes requires protective protein/cathepsin A.

Human lysosomal N-acetyl-alpha-neuraminidase is deficient in two lysosomal storage disorders, sialidosis, caused by structural mutations in the neuraminidase gene, and galactosialidosis, in which a primary defect of protective protein/cathepsin A (PPCA) leads to a combined deficiency of neuraminidase and beta-D-galactosidase. These three glycoproteins can be isolated in a high molecular weight multi-enzyme complex, and the enzymatic activity of neuraminidase is contingent on its interaction with PPCA. To explain the unusual need of neuraminidase for an auxiliary protein, we examined, in transfected COS-1 cells, the effect of PPCA expression on post-translational modification, turnover and intracellular localization of neuraminidase. In pulse-chase studies, we show that the enzyme is synthesized as a 46 kDa glycoprotein, which is poorly phosphorylated, does not undergo major proteolytic processing and is secreted. Importantly, its half-life is not altered by the presence of PPCA. However, neuraminidase associates with the PPCA precursor shortly after synthesis, since the latter protein co-precipitates with neuraminidase using anti-neuraminidase antibodies. We further demonstrate by subcellular fractionation of transfected cells that neuraminidase segregates to mature lysosomes only when accompanied by wild-type PPCA, but not by transport-impaired PPCA mutants. These data suggest a novel role for PPCA in the activation of lysosomal neuraminidase, that of an intracellular transport protein.     

The presence of a reduced amount of 32-kd “protective” protein is a distinct biochemical finding in late infantile galactosialidosis

Summary The biochemical defect underlying the late infantile form of galactosialidosis has been investigated in fibroblasts from two patients presenting with this phenotype. Immunoprecipitation experiments demonstrated that a reduced amount of 32-kd protective protein and a normal amount of its precursor are present in late infantile galactosialidosis fibroblasts, while neither of the two polypeptides are detectable in early infantile and juvenile/adult fibroblasts. Leupeptin treatment led to a slight increase in the amount of 54-kd and 32-kd polypeptides in both late-infantile galactosialidosis cell lines. Uptake studies in one of the two cell lines confirmed the hypothesis that a block in the maturation of the protective protein is responsible for the late infantile type of galactosialidosis. This mutation seems to be a distinct finding in all patients affected by this form of the disease.     

Identification and in vitro reconstitution of lysosomal neuraminidase from human placenta

Lysosomal neuraminidase from human placenta has been obtained in its active form by association of an inactive neuraminidase polypeptide with beta-galactosidase and the protective protein. Using a specific antiserum, we have now identified a 66-kDa protein as the inactive neuraminidase polypeptide. It is specifically recognized on immunoblots only in its nonreduced state, and it coprecipitates with neuraminidase activity. The 66-kDa polypeptide is substantially glycosylated (38-kDa protein core with 7-14 N-linked oligosaccharide chains), a feature characteristic of lysosomal integral membrane proteins. Specific removal of the 66-kDa neuraminidase polypeptide from glycoprotein preparations prevents the generation of neuraminidase activity. Removal of beta-galactosidase or destruction of the protective protein also hinders the formation of active neuraminidase. Reconstitution of neuraminidase activity is observed after mixing glycoprotein preparations, depleted in different components of the beta-galactosidase-neuraminidase-protective protein complex, indicating that all three components of the complex are required for neuraminidase activity. Association of the neuraminidase polypeptide and the protective protein generates unstable neuraminidase activity, whereas association with beta-galactosidase is required for stability.     

A mutation in a mild form of galactosialidosis impairs dimerization of the protective protein and renders it unstable.

The lysosomal disorder galactosialidosis is caused by deficiency of the protective protein in the absence of which the activities of the enzymes beta-galactosidase and neuraminidase are reduced. Aside from its protective function towards the two glycosidases, this protein has cathepsin A-like activity. A point mutation in the protective protein gene, resulting in the substitution of Phe412 with Val in the gene product, was identified in two unrelated patients with the late infantile form of the disease. Expression in COS-1 cells of a protective protein cDNA with the base substitution resulted in the synthesis of a mutant protein that lacks cathepsin A-like activity. The newly made mutant precursor was shown to be partially retained in the endoplasmic reticulum. Only a fraction is transported to the lysosomes where it is degraded soon after proteolytic processing into the mature two-chain form. Since the mutant precursor, contrary to the wild type protein, does not form homodimers, the dimerization process might be a condition for the proper targeting and stable conformation of the protective protein. These results clarify the mechanism underlying the combined deficiency in these patients, and give new insight into the structure-function relationship of the wild type protein.     

Human lysosomal protective protein has cathepsin A-like activity distinct from its protective function

The protective protein was first discovered because of its deficiency in the metabolic storage disorder galactosialidosis. It associates with lysosomal beta-galactosidase and neuraminidase, toward which it exerts a protective function necessary for their stability and activity. Human and mouse protective proteins are homologous to yeast and plant serine carboxypeptidases. Here, we provide evidence that this protein has enzymatic activity similar to that of lysosomal cathepsin A: 1) overexpression of human and mouse protective proteins in COS-1 cells induces a 3-4-fold increase of cathepsin A-like activity; 2) this activity is reduced to approximately 1% in three galactosialidosis patients with different clinical phenotypes; 3) monospecific antibodies raised against human protective protein precipitate virtually all cathepsin A-like activity in normal human fibroblast extracts. Mutagenesis of the serine and histidine active site residues abolishes the enzymatic activity of the respective mutant protective proteins. These mutants, however, behave as the wild-type protein with regard to intracellular routing, processing, and secretion. In contrast, modification of the very conserved Cys60 residue interferes with the correct folding of the precursor polypeptide and, hence, its intracellular transport and processing. The secreted active site mutant precursors, endocytosed by galactosialidosis fibroblasts, restore beta-galactosidase and neuraminidase activities as effectively as wild-type protective protein. These findings indicate that the catalytic activity and protective function of the protective protein are distinct.     

Purification and partial characterization of lysosomal neuraminidase from human placenta

Lysosomal neuraminidase and beta-galactosidase are present in a complex together with a 32-kDa protective protein. This complex has been purified and the different components have been dissociated using potassium isothiocyanate (KSCN) treatment. beta-Galactosidase remains catalytically active, but neuraminidase loses its activity upon dissociation. The inactive dissociated neuraminidase was purified by removing the remaining non-dissociated beta-galactosidase/protective protein complex using beta-galactosidase-specific affinity chromatography. The dissociated neuraminidase material shows two major polypeptides on SDS-PAGE with an apparent molecular mass of 76 kDa and 66 kDa. Subsequently the 32-kDa protective protein was dissociated from the beta-galactosidase/protective protein complex, and purified. Antibodies raised against the dissociated inactive neuraminidase preparation specifically immunoprecipitate the active neuraminidase present in the complex with beta-galactosidase and protective protein. By immunoblotting evidence is provided that the 76-kDa protein is a subunit of neuraminidase which, in association with the 32-kDa protective protein, is essential for neuraminidase activity.     

Characterization of a unique glycoprotein antigen expressed on the surface of human neuroblastoma cells

In order to develop a molecular probe to delineate chemical and biological characteristics of human neuroblastoma cells, a murine monoclonal antibody (Mab 5G3) was produced that is directed to a glycoprotein, preferentially expressed on the surface of such cells. This antibody is of IgG2a isotype, has an association constant of 8 X 10(9) M-1, and reacts preferentially with human neuroblastoma cell lines and fresh frozen tissue sections in enzyme-linked immunosorbent assay and immunoperoxidase assays, respectively. Minimal reactivity is observed with a variety of lymphoblastoid cell lines and normal fetal and adult tissues. Mab 5G3 specifically recognizes a neuroblastoma target glycoprotein antigen of 215 kDa that is derived from a 200-kDa precursor, as evident from pulse-chase biosynthetic studies. Treatment with tunicamycin revealed that both molecules contain N-asparagine-linked oligosaccharides; however, only the 215-kDa species is resistant to treatment with endo-beta-N-acetylglucosaminidase H and sensitive to neuraminidase, indicating that it contains trimmed and terminally sialylated oligosaccharides of the "complex" type. In contrast, the 200-kDa precursor is sensitive to endo-beta-N-acetylglucosaminidase H and resistant to neuraminidase treatment indicating that it contains high-mannose non-processed oligosaccharides. The 215-kDa molecule is sulfated, phosphorylated at serine residues, and expressed on the cell surface. A molecule of 200 kDa is detected by Mab 5G3 in spent culture medium of human neuroblastoma cells which is neither sulfated nor phosphorylated.     

Processing of lysosomal beta-galactosidase. The C-terminal precursor fragment is an essential domain of the mature enzyme

Lysosomal beta-D-galactosidase (beta-gal), the enzyme deficient in the autosomal recessive disorders G(M1) gangliosidosis and Morquio B, is synthesized as an 85-kDa precursor that is C-terminally processed into a 64-66-kDa mature form. The released approximately 20-kDa proteolytic fragment was thought to be degraded. We now present evidence that it remains associated to the 64-kDa chain after partial proteolysis of the precursor. This polypeptide was found to copurify with beta-gal and protective protein/cathepsin A from mouse liver and Madin-Darby bovine kidney cells and was immunoprecipitated from human fibroblasts but not from fibroblasts of a G(M1) gangliosidosis and a galactosialidosis patient. Uptake of wild-type protective protein/cathepsin A by galactosialidosis fibroblasts resulted in a significant increase of mature and active beta-gal and its C-terminal fragment. Expression in COS-1 cells of mutant cDNAs encoding either the N-terminal or the C-terminal domain of beta-gal resulted in the synthesis of correctly sized polypeptides without catalytic activity. Only when co-expressed, the two subunits associate and become catalytically active. Our results suggest that the C terminus of beta-gal is an essential domain of the catalytically active enzyme and provide evidence that lysosomal beta-galactosidase is a two-subunit molecule. These data may give new significance to mutations in G(M1) gangliosidosis patients found in the C-terminal part of the molecule.     

Generation of truncated forms of the NG2 proteoglycan by cell surface proteolysis.

NG2 is a chondroitin sulfate proteoglycan that is expressed on dividing progenitor cells of several lineages including glia, muscle, and cartilage. It is an integral membrane proteoglycan with a core glycoprotein of 300 kDa. In the present study we have characterized three molecular forms of the NG2 core protein expressed by different cell lines. Many cell lines that express the full length 300-kDa NG2 core protein also release a 290-kDa form into the medium. This species lacks the cytoplasmic domain but contains almost the entire ectodomain. Two core protein species, the intact 300-kDa form and a truncated 275-kDa form, are expressed at the surface of an NG2-transfected cell line U251NG52. The 275-kDa species lacks the cytoplasmic domain and at least 64 amino acids of the ectodomain. Mild trypsinization of B49 cells also generates the 275-kDa species, suggesting that this component is produced by proteolysis of the 300-kDa form. Conversion of the 300-kDa species to the 275-kDa form in U251NG52 cells is stimulated by reagents such as phorbol esters, which activate protein kinase C. Phorbol esters are also known to induce expression of metalloproteinases such as collagenase and stromelysin, which could be responsible for cleavage of the 300-kDa core protein. Although B49 cells do not spontaneously produce the truncated 275-kDa species, use of monoclonal antibodies against NG2 to block the interaction between NG2 and type VI collagen results in the appearance of the 275-kDa component in these cells. Thus the interaction between NG2 and type VI collagen, which contains a Kunitz-type proteinase inhibitor sequence in the alpha 3 chain, may protect the proteoglycan against proteolysis. This is consistent with the observed deficiency of U251NG52 cells in anchoring type VI collagen at the surface.     

Cell-free sulfation of the contact site A glycoprotein of Dictyostelium discoideum and of a partially glycosylated precursor

An 80-kDa glycoprotein of Dictyostelium discoideum, designated contact site A, has been implicated in EDTA-stable cell adhesion. This protein is known to be the major sulfated protein of aggregation-competent cells and has been shown to contain two types of carbohydrate, sulfated type 1 and unsulfated type 2 carbohydrate moieties. Here we investigate the cell-free sulfation of this protein. In the homogenate of developing cells, [35S]sulfate was transferred by endogenous sulfotransferase from [35S]3'-phosphoadenosine-5'-phosphosulfate to the contact site A glycoprotein and to various other endogenous proteins. The sulfate was transferred to carbohydrate rather than to tyrosine residues. After differential centrifugation of the homogenate, the capacity for sulfation of the contact site A glycoprotein was barely detected in the plasma membrane-enriched 10,000 X g pellet fraction which contained the bulk of this glycoprotein, but was largely recovered in the 100,000 X g pellet fraction which contained only a small portion of this glycoprotein. After sucrose gradient centrifugation, the membranes containing the sulfation capacity were found to have a density characteristic for Golgi membranes. In immunoblots, monoclonal antibodies raised against the contact site A glycoprotein recognized not only this 80-kDa protein, but also a sulfatable 68-kDa protein found in the 100,000 X g pellet fraction. The 68-kDa protein did not react with monoclonal antibodies against type 2 carbohydrate but was converted by endoglycosidases F and H into a 53-kDa protein, indicating that it was a partially glycosylated form of the 80-kDa glycoprotein containing only type 1 carbohydrate. Isoelectric focusing showed that a substantial portion of the 68-kDa glycoprotein was unsulfated, even after cell-free sulfation. The 68-kDa glycoprotein was not found in the plasma membrane-enriched 10,000 X g pellet fraction and did not accumulate in parallel with the 80-kDa contact site A glycoprotein during cell development. We conclude that the 68-kDa glycoprotein is a precursor that is converted by attachment of type 2 carbohydrate and sulfation of type 1 carbohydrate into the mature 80-kDa glycoprotein. The precursor nature of the 68-kDa glycoprotein was supported by results obtained with mutant HL220 which is defective in glycosylation (Murray, B. A., Wheeler, S., Jongens, T., and Loomis, W. F. (1984) Mol. Cell. Biol. 4, 514-519). This mutant specifically lacks type 2 carbohydrate and produces a 68-Kda glycoprotein instead of the 80-kDa contact site A glycoprotein (Yoshida, M., Stadler, J., Bertholdt, G., and Gerisch, G. (1984) EMBO J. 3, 2663-2670).(ABSTRACT TRUNCATED AT 400 WORDS)     

Biosynthesis and processing of the mannose receptor in human macrophages

The biosynthesis and processing of the human mannose receptor has been studied in monocyte-derived macrophages. Adherent cells were labeled for 60 min with Trans35S (a mixture of 35S-labeled methionine and cysteine), chased, and subjected to immunoprecipitation by antibody raised against the human placental receptor. The antibody immunoprecipitated a single protein of molecular mass 162 kDa; precipitation of the labeled receptor could be inhibited by placental receptor. The results presented demonstrate that the receptor is synthesized as a 154-kDa precursor which is processed to 162 kDa in 90 min. The precursor is a glycoprotein bearing endoglycosidase H-sensitive oligosaccharides; the 162-kDa form is endoglycosidase H-resistant but peptide:N-glycanase-sensitive. Desialylation of the mannose receptor with neuraminidase generates a protein which is recognized by peanut agglutinin, a lectin that specifically binds desialylated O-linked oligosaccharides. Thus, the human macrophage mannose receptor bears both N- and O-linked oligosaccharide chains. Newly synthesized mannose receptor exhibits a half-life of 33 h as determined by pulse-chase studies. This indicates that on the average, each molecule of receptor recycles between the cell surface and endosomes hundreds of times before degradation.     

Stability and covalent modification of P-glycoprotein in multidrug-resistant KB cells

An antipeptide antibody (P7) to P-glycoprotein has been produced by immunizing rabbits with a synthetic peptide. Antibody P7 is directed against the amino-terminal region of P170 (residues 28-35). The antibody immunoprecipitates a 170-kDa P-glycoprotein from extracts of drug-resistant KB-V1 cells that is not present in the drug-sensitive cell line KB-3-1. Antibody P7 was used to quantitate the amount of P-glycoprotein present in drug-resistant KB lines at various levels of resistance and to demonstrate the presence of P-glycoprotein in NIH 3T3 cells transfected with a cloned MDR1 cDNA or human genomic DNA encoding MDR1. Pulse-chase labeling experiments demonstrated that P-glycoprotein is synthesized as a 140-kDa precursor which is slowly converted over 2-4 h to a 170-kDa glycoprotein. Tunicamycin treatment blocks the conversion of the precursor to the mature form, and removal of N-linked oligosaccharides with Endo F reduces the relative molecular weight of P-glycoprotein to 140K. The mobility of mature P-glycoprotein is unaffected by treatment with neuraminidase and Endo H. These data indicate that P-glycoprotein is N-glycosylated and contains little or no neuraminic acid. P-Glycoprotein is also phosphorylated, and the extent of phosphate incorporated is proportional to the amount of protein present in drug-resistant cells.     

Two-step glycosylation of the contact site A protein of Dictyostelium discoideum and transport of an incompletely glycosylated form to the cell surface

Two different types of oligosaccharides, designated type 1 and 2 carbohydrate residues, are present on the contact site A molecule, an 80-kDa glycoprotein involved in the formation of EDTA-stable cell adhesion during cell aggregation in Dictyostelium discoideum. The first precursor detected by pulse-chase labeling with [35S]methionine was a 68-kDa glycoprotein carrying type 1 carbohydrate. Conversion of the precursor into the 80-kDa form occurred simultaneously with the addition of type 2 carbohydrate. Tunicamycin inhibited type 1 glycosylation more efficiently than type 2 glycosylation. The first precursor detected in tunicamycin-treated cells by pulse-chase labeling was a 53-kDa protein lacking both carbohydrates, which was converted through addition of type 2 carbohydrate into a 66-kDa final product. Labeling of intact cells indicated that this 66-kDa glycoprotein is transported to the cell surface. Prolonged treatment with tunicamycin resulted in the accumulation within the cells of the 53-kDa precursor with no detectable exposure of this protein on the cell surface. It is concluded that type 1 carbohydrate, which is cotranslationally added in N-glycosidic linkages, is neither required for transport of the protein to the Golgi apparatus nor for type 2 glycosylation or protection of the protein against proteolytic degradation. Incapability of tunicamycin-treated cells of forming EDTA-stable cell contacts suggests a role for type 1 carbohydrate in cell adhesion. Type 2 carbohydrate is added posttranslationally. It is required in the absence of type 1 glycosylation for transport of the protein to the cell surface.